PCR method and related apparatus

ABSTRACT

A method for balancing multiplexed PCR methods is provided. In the method, two or more sequential temporal PCR stages are used to effectively separate two or more PCR reactions in a single tube as an alternative to primer limiting to modulate the relative rate of production of a first amplicon by a first primer set and a second amplicon by a second primer set during the first and second amplification stages. Also provided are rapid RT-PCR methods that find particular use in intraoperative diagnoses and prognoses, for instance in diagnosing malignant esophageal adenocarcenoma by determining expression levels of carcinoembryonic antigen (CEA) in sentinel lymph nodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 to U.S. patentapplication Ser. No. 10/090,326, filed Mar. 4, 2002, which, in turn,claims priority under 35 U.S.C. § 119(e) to U.S. Application Ser. No.60/273,277, filed Mar. 2, 2001, both of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERAL SUPPORT

This invention was made with government support under Grant No.CA90665-01, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND

This Application discloses a rapid PCR method, with particular focus ona rapid multiplex QRT-PCR method and related compositions and apparatus.

Polymerase chain reaction (PCR) is a powerful tool in the field ofmolecular biology. This technique allows for replicating/amplifyingtrace amounts DNA fragments into quantities that can be analyzed in ameaningful way. As such this technology has been adapted to molecularbiological applications like DNA sequencing, DNA fingerprinting, etc.Additionally, this method has the ability to detect specific DNAfragments in samples, whose presence may reflect a pathological state.Therefore, this method is finding new applications in the field ofmolecular diagnostics. Furthermore with the development of real-timequantitative PCR (QPCR), this technology has become more reliable aswell as amenable to automation. Currently, this technology is used forthe detection of viral and bacterial pathogens in clinical samples andfor the detection of cancer cells in patients with a history ofleukemias (and other cancers such as those that arise in the breast,lung, colon, esophagus and skin).

PCR in molecular diagnostics, despite its advantages, has severalshortcomings. Often, this is a technique that is dependent on thetechnical expertise of the operator. Additionally, it is also very proneto contamination. The combination of these two above-mentioned factorsresults in false negative and false positive results respectively.Therefore, there is a need to incorporate internal controls along withthe target of interest as well as automate the process to ensureoperation within a closed system (to eliminate contamination). Theincorporation of controls involves the amplification of severaldifferent sets of DNA fragments in addition to the target of interest.These controls provide information about the quality of the samplesbeing analyzed as well as that of the assay in any given run. Fortechnical reasons, analyzing multiple DNA targets within a sample in thesame reaction tube through PCR (known as multiplexing) does not workwell when the targets are not present in similar abundance at thebeginning of the reaction. Historically, investigators have attempted toovercome this by limiting the primers in the PCR reaction. This approachis based on the idea that by limiting the reagents in one reaction, itstops the reaction at a point after adequate amplification has occurredfor that target but before inhibition of amplification of othersequences. Though this method can increase the difference in initialabundance between the two targets that will still result in thesuccessful amplification of both targets, it does not allow for thedetection of a rare target in the milieu of a second target that isseveral orders of magnitude more abundant. Furthermore, when a rapidQPCR assay is called for, decreasing the primer concentration alsoworsens quantitation.

A further limitation of current PCR technologies is the time it takes toperform PCR diagnoses. Typical PCR reactions take hours, not minutes. Asdescribed below, decreasing the time it takes to carry out a PCRreaction is desirable for many reasons. Therefore, there is a need foran automated PCR based point of care molecular diagnostic system,especially a rapid, multiplexed (RT-)PCR assay.

SUMMARY OF THE INVENTION

Rapid and robust PCR and RT-PCR methods are provided that permitexecution of a complete PCR reaction in minutes, not hours, permittinguse of PCR in intraoperative diagnoses, for example, but withoutlimitation, for detecting micrometastases in sentinel lymph nodes, as ansuperior alternative to, or in addition to typical pathological methodssuch as histopathological examination of lymph nodes.

Also provided is an alternative to primer limiting as a method forbalancing a multiplex PCR reaction, especially quantitative PCRamplifications, with particular usefulness in QRT-PCR reactions. Thismethod finds particular use when one target sequence to be amplified isfar less prevalent than another to be amplified in the same reactionmixture. The method comprises the step of conducting a PCR amplificationon a DNA sample in a PCR reaction mixture in a first amplification stageand a second amplification stage. The PCR amplification of the secondamplification stage is conducted under different reaction conditionsthan the PCR amplification of the first amplification stage to modulatethe relative rate of production of a first amplicon by a first primerset and a second amplicon by a second primer set during the first andsecond amplification stages. Additional amplification stages may beadded.

Two non-limiting specific embodiments of this method are disclosed. Inthe first embodiment, the second primer set is added to the reactionmixture at the beginning of the second amplification stage, therebylimiting the physical presence of the second primer set during the firststage. In this method, the rarer target sequence preferably is amplifiedbefore the less-rare sequence which typically is a control, such asβ-gus or 18SrRNA sequences.

In the second embodiment, the PCR reaction mixture includes the firstprimer set having a first effective Tm and the second primer set havinga second effective Tm different from the first effective Tm. Therelative rate of production of the first amplicon by the first primerset and the second amplicon by the second primer set during the firstand second amplification stages is modulated by conducting the annealingstep of the first amplification stage at a different temperature thanthe annealing step of the second amplification stage. In this secondembodiment, the annealing temperature for the second amplification stagemay be higher or lower than the annealing temperature for the firstamplification stage.

Also provided is a rapid RT-PCR method that is based upon the findingthat the reverse transcription reaction of the RT-PCR method need not beperformed for longer than about 10 minutes, and preferably only forabout two minutes. This rapid step, when coupled with a rapid PCRprocedure conducted sequentially with the RT reaction in the samereaction vessel as the RT reaction, permits intraoperative use of theRT-PCR reaction, especially when the entire process is automated.

Each of the above-described PCR and RT-PCR process find special utilityin their use in quantitative PCR methods, such as QPCR and QRT-PCR,which are typically monitored during the PCR amplification by theaccumulation of, or loss of, a fluorescent reporter, for instance by theuse of TAQMAN and molecular beacon probes.

The above-described methods may be automated in a cartridge-basedsystem, thereby reducing human error in the methods, as well as thepotential for contamination. In an automated system, reagents for thevarious reactions are added sequentially according to a programmedsequence. A cartridge, for use in an automated system, for performingthe described methods also is provided.

Also provided are specific uses for the rapid PCR methods describedherein. In one embodiment, an intraoperative PCR diagnostic method isprovided that includes the steps of: obtaining a tissue sample from apatient in an operation; analyzing the sample according to one of theabove-described PCR methods; determining if expression of an indicatortranscript exceeds a threshold level; and continuing the operation in amanner dictated by results of the analyzing step. In another embodiment,a method for rapid detection of a malignancy is provided that includesthe steps of: obtaining nucleic acid from a tumor biopsy; performing aPCR method specific to an indicator transcript on the nucleic acidaccording to one of the above-described PCR methods; and determining ifexpression of the indicator transcript exceeds a threshold level,thereby indicating a malignancy.

In an additional embodiment, a method for rapid detection ofmetastasized adenocarcenoma of the esophagus is provided. The methodincludes the steps of: obtaining RNA from a sentinel lymph node;performing a quantitative RT-PCR method specific to CEA on the RNAaccording to any one of the above-described PCR methods; and determiningif expression of CEA exceeds a threshold level.

Lastly, also provided are specific novel oligonucleotide primers usefulin the detection of sequences specific to CEA and tyrosinase genes, aswell as β-gus and 18SrRNA sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a cartridge according to one embodimentof the present invention.

FIG. 2 shows two graphs comparing the sensitivity of the one-tube RT-PCRwith (B) and without (A) a wax separating the RT and PCR reactions.

FIG. 3 is a photograph of an ethidium bromide-stained gel comparingRT-PCR product prepared with and without use of a wax layer to separatethe RT and PCR reactions.

FIG. 4 provides four graphs showing the amplification of CEA and 18SrRNAin a multiplexed reaction as described in Example 3. Panels A and C showthe results for 18S rRNA and CEA respectively when run in a singleplexusing optimal conditions. Panels B and D show 18S rRNA and CEAmultiplexed.

FIG. 5 provides the CEA nucleotide sequence, GenBank Accession No.XM_(—)13012777 (SEQ ID NO: 1).

FIG. 6 is a graph showing the relative CEA expression measured in 10esophageal tumors (light gray), 4 histologically positive (N₁) lymphnodes (white), and 10 benign lymph nodes from patients without cancer(dark gray).

FIG. 7 shows the Relative CEA expression measured in histologicallynegative lymph nodes from 30 esophageal cancer patients. Graph shows thehighest CEA level found for each patient. Patients 1-20 (dark graycolumns) did not recur, whereas patients 21-30 (light gray columns) didrecur. The dotted line indicates the most accurate cutoff value forpredicting recurrence. Using this cutoff, QRT-PCR correctly classified90% of patients with respect to disease recurrence at 3 years.

FIGS. 8A-D are Kaplan-Meier survival curves for disease-free survivalfor patients classified by RT-PCR (8A and 8B) and QRT-PCR (8C and 8D).Although log-rank tests indicate that both predictors are statisticallysignificant, the superior specificity of QRT-PCR compared with RT-PCRleads to a greater ability to differentiate patients on the basis oftheir risk of recurrence (P=0.0038 for RT-PCR and <0.0001 for QRT-PCR).

FIG. 9 shows a SmartCycler analysis of CEA expression in formalin-fixedlymph nodes from 30 pathologically node negative esophagus cancerpatients. Gray bars (21-30) indicate patients who suffered recurrence,black bars (1-20) indicate patients who did not suffer recurrence. Lymphnodes with no detectable CEA expression have been arbitrarily plotted ata level of 0.02. The dotted line indicates the most accurate CEAexpression cut-off value (0.183) for predicting disease recurrence.

FIG. 10 shows a Kaplan-Meier disease free survival curve for 30 pN₀esophagus cancer patients stratified as LN positive or negative (aboveor below the cut-off value of 0.183) based on Smartcycler QRT-PCR.

FIG. 11 shows Smartcycler analysis of CEA expression in frozen lymphnodes. White (1-11)=nodes from patients without cancer; black(12-17)=nodes determined on final pathology to be negative for disease;gray (20-23)=nodes found to be positive for disease; gray (18 and19)=nodes that were negative by intra-operative frozen section butpositive on fixed tissue histology; star=highly expressing node from anode negative patient who suffered disease recurrence. The value of0.001 was arbitrarily assigned to all samples that had no detectableexpression of CEA.

FIGS. 12A-C show the optimization of the rapid QRT-PCR assay for β-gusand CEA. FIG. 12A compares the Ct values for assays with differentdenaturation times and a 30 second extension time. FIG. 12B compares theeffect of different extension times on the Ct value when thedenaturation time is held constant at 10 seconds. FIG. 12C demonstratesthe effect of different RT times when the PCR conditions are constant.

FIG. 13 shows Ct values for β-gus and CEA in a 8-fold serial dilution ofCEA in a constant background of β-gus. This figure compares the slowconventional PCR (10 sec denaturation followed by a 30 secannealing/extension step) with rapid PCR (1 sec denaturation followed bya 6 second annealing/extension step). ♦=10, 30 GUS; □=1, 6 GUS; Δ=10, 30CEA; and ●=1, 6 CEA.

FIG. 14 is a RT-PCR run time comparison between a 15 min (2 min RT and ⅓PCR), 17 min (2 min RT and ⅙ PCR), 20 min (5 min RT, ⅙ PCR) and a 38 min(10 min RT and 5/15 PCR) assays.

FIG. 15 shows four amplification plots for CEA singleplex (panel A), CEAin the temperature-controlled multiplex (panel B), CEA in the ordinarymultiplex (panel C) and CEA in the conventional primer limitingmultiplex (panel D)

FIG. 16 shows a comparison of the singleplex PCR for CEA with thetemperature-controlled multiplex, ordinary multiplex and theconventional primer limiting multiplex. The delta Ct values for a serialdilution of CEA in constant background of β-gus are compared. Delta Ctof 25 is used to denote a dilution point that failed to amplify.

FIG. 17 is a histogram of CEA expression relative to β-gus in lymphnodes. Bars in Black (1-3) correspond to lymph nodes from patients withcancer with histological evidence of cancer. Bars in Gray (4-8)correspond to lymph nodes from patients without cancer. Samples 4 and 6had no detectable CEA expression.

FIG. 18 shows four representative amplification plots for samples withthe expression of CEA (A) and tyrosinase (C), and samples without theexpression of CEA (B) and tyrosinase (D). In panels A and B, thefluorescence curves for β-gus (+), CEA (⋄) and CEA IC (−) are shown.Similarly in panels C and D, the fluorescence curves for β-gus (⋄),tyrosinase (−) and tyrosinase IC (*) are shown.

DETAILED DESCRIPTION

Provided are improved PCR methods that permit rapid cycling and/orimproved sensitivity for PCR-based molecular diagnostics, especiallywith respect to quantitative PCR methods, including QRT-PCR. Theseimproved methods permit PCR to be used intraoperatively, and also areuseful in detecting rare nucleic acid species, even in multiplexed PCRreactions that concurrently detect a more prevalent control nucleicacid.

A typical PCR reaction includes multiple amplification steps, or cyclesthat selectively amplify a target nucleic acid species. A fulldescription of the PCR process, and common variations thereof, such asquantitative PCR (QPCR), real-time QPCR, reverse transcription PCR(RT-PCR) and quantitative reverse transcription PCR (QRT-PCR) is beyondthe scope of this disclosure and these methods are well-described in theart and have been broadly commercialized. A typical PCR reactionincludes three steps: a denaturing step in which a target nucleic acidis denatured; an annealing step in which a set of PCR primers (forwardand backward primers) anneal to complementary DNA strands; and anelongation step in which a thermostable DNA polymerase elongates theprimers. By repeating this step multiple times, a DNA fragment isamplified to produce an amplicon, corresponding to the target DNAsequence. Typical PCR reactions include 30 or more cycles ofdenaturation, annealing and elongation. In many cases, the annealing andelongation steps can be performed concurrently, in which case the cyclecontains only two steps.

The lengths of the denaturation, annealing and elongation stages may beany desirable length of time. However, in attempting to shorten the PCRamplification reaction to a time suitable for intraoperative diagnosis,it has been found that the lengths of these steps can be in the secondsrange, rather than the minutes range. Specifically, with certain newthermal cyclers being capable of generating a thermal ramp rate (ΔT) ofat least about 5° C. per second, PCR amplifications in 20 minutes arepossible. As used herein, the times provided for each step of the PCRcycle does not include ramp times. The denaturation step may beconducted for times of one second or less. In fact, some thermal cyclersdo not have settings for “0 seconds” which may be the optimal durationof the denaturation step. That is, it is enough that the thermal cyclerreaches the denaturation temperature. The annealing and elongation stepsare optimally less than 10 seconds each, and when conducted at the sametemperature, the combination annealing/elongation step may be less than10 seconds.

As described herein, each cycle may be shortened considerably withoutsubstantial deterioration of production of amplicons by usingsubstantially increased primer concentrations, typically greater thanabout 400 nM, and often greater than about 800 nM, though the optimalconcentration of primers will vary somewhat from assay-to-assay.Sensitivity of RT-PCR assays may be enhanced by the use of a sensitiveReverse Transcriptase enzyme (described herein) and/or highconcentrations of Reverse Transcriptase primer to produce the initialtarget PCR template.

The specificity of any given PCR reaction relies heavily, but notexclusively, on the identity of the primer sets. The primer sets arepairs of forward and reverse oligonucleotide primers that anneal to atarget DNA sequence to permit amplification of the target sequence,thereby producing a target sequence-specific amplicon. As used herein, a“derivative” of a specified oligonucleotide is an oligonucleotide thatbinds to the same target sequence as the specified oligonucleotide andamplifies the same target sequence to produce essentially the sameamplicon as the specified oligonucleotide but for differences betweenthe specified oligonucleotide and the derivative. The derivative maydiffer from the specified oligonucleotide by insertion, deletion and/orsubstitution of any residue of the specified sequence so long as thederivative substantially retains the characteristics of the specifiedsequence in its use for the same purpose as the specified sequence.

As used herein, “reagents” for any enzymatic reaction mixture, such as areverse transcription and PCR reaction mixture, are any compound orcomposition that is added to the reaction mixture including, withoutlimitation, enzyme(s), nucleotides or analogs thereof, primers andprimer sets, buffers, salts and co-factors. As used herein, unlessexpressed otherwise, “reaction mixture” includes all necessary compoundsand/or compositions necessary to perform that enzymatic reaction, evenif those compounds or compositions are not expressly indicated.

Multiplexed PCR assays may be optimized, or balanced, by time-shiftingthe production of amplicons, rather than by manipulating primerconcentrations. This may be achieved by using two primer sets, eachprimer set having a different Tm so that a two-stage PCR assay can beperformed, with different annealing and/or elongation temperatures foreach stage to favor the production of one amplicon over the other. Thistime and temperature shifting method permits optimal balancing of themultiplex reaction without the difficulties faced when manipulation ofprimer concentrations is used to balance the reaction. This technique isespecially useful in a multiplex reaction where it is desirable toamplify a rare cDNA along with a control cDNA, such as the CEA/β-GUSexample shown below.

A quantitative reverse transcriptase polymerase chain reaction (QRT-PCR)method is provided for rapidly and accurately detecting low abundanceRNA species in a population of RNA molecules (for example, and withoutlimitation, total RNA or mRNA), including the steps of: a) incubating anRNA sample with a reverse transcriptase and a high concentration of atarget sequence-specific reverse transcriptase primer under conditionssuitable to generate cDNA; b) subsequently adding suitable polymerasechain reaction (PCR) reagents to the reverse transcriptase reaction,including a high concentration of a PCR primer set specific to the cDNAand a thermostable DNA polymerase to the reverse transcriptase reaction,and c) cycling the PCR reaction for a desired number of cycles and undersuitable conditions to generate PCR product (“amplicons”) specific tothe cDNA. By temporally separating the reverse transcriptase and the PCRreactions, and by using reverse transcriptase-optimized andPCR-optimized primers, excellent specificity is obtained. The reactionis conducted in a single tube (all tubes, containers, vials, cells andthe like in which a reaction is performed may be referred to herein,from time to time, generically, as a “reaction vessel”), removing asource of contamination typically found in two-tube reactions. The highconcentration primers permit very rapid QRT-PCR reactions, typically onthe order of 20 minutes from the beginning of the reverse transcriptasereaction to the end of a 40 cycle PCR reaction. The realization of sucha rapid QRT-PCR experiment is assisted by the availability of thermalcycling devices capable of generating a thermal ramp rate (ΔT) of atleast about 5° C. per second.

The reaction c) may be performed in the same tube as the reversetranscriptase reaction by adding sufficient reagents to the reversetranscriptase (RT) reaction to create good, or even optimal conditionsfor the PCR reaction to proceed. A single tube may be loaded, prior tothe running of the reverse transcriptase reaction, with: 1) the reversetranscriptase reaction mixture, and 2) the PCR reaction mixture to bemixed with the cDNA mixture after the reverse transcriptase reaction iscompleted. The reverse transcriptase reaction mixture and the PCRreaction mixture may be physically separated by a solid, or semi-solid(including amorphous, glassy substances and waxy) barrier of acomposition that melts at a temperature greater than the incubationtemperature of the reverse transcriptase reaction, but below thedenaturing temperature of the PCR reaction. The barrier composition maybe hydrophobic in nature and forms a second phase with the RT and PCRreaction mixtures when in liquid form. One example of such a barriercomposition is wax beads, commonly used in PCR reactions, such as theAMPLIWAX PCR GEM products commercially available from Applied Biosystemsof Foster City, Calif. and the STRATASPHERE Magnesium Wax Beads,commercially available from Stratagene of La Jolla, Calif.

Alternatively, the separation of the reverse transcriptase and the PCRreactions may be achieved by adding the PCR reagents, including the PCRprimer set and thermostable DNA polymerase, after the reversetranscriptase reaction is completed. Preferably the PCR reagents, areadded mechanically by a robotic or fluidic means to make samplecontamination less likely and to remove human error.

The products of the QRT-PCR process may be compared after a fixed numberof PCR cycles to determine the relative quantity of the RNA species ascompared to a given reporter gene. One method of comparing the relativequantities of the products of the QRT-PCR process is by gelelectrophoresis, for instance, by running the samples on a gel anddetecting those samples by one of a number of known methods including,without limitation, Southern blotting and subsequent detection with alabeled probe, staining with ethidium bromide and incorporatingfluorescent or radioactive tags in the amplicons.

However, the progress of the PCR reaction typically is monitored byanalyzing the relative rates of amplicon production for each PCR primerset. Monitoring amplicons production may be achieved by a number ofprocesses, including without limitation, fluorescent primers,fluorogenic probes and fluorescent dyes that bind double-stranded DNA. Acommon method is the fluorescent 5′ nuclease assay. This method exploitsthe 5′ nuclease activity of certain thermostable DNA polymerases (suchas Taq or Tfl DNA polymerases) to cleave an oligomeric probe during thePCR process. The oligomer is selected to anneal to the amplified targetsequence under elongation conditions. The probe typically has afluorescent reporter on its 5′ end and a fluorescent quencher of thereporter at the 3′ end. So long as the oligomer is intact, thefluorescent signal from the reporter is quenched. However, when theoligomer is digested during the elongation process, the fluorescentreporter is no longer in proximity to the quencher. The relativeaccumulation of free fluorescent reporter for a given amplicon may becompared to the accumulation of the same amplicons for a control sampleand/or to that of a control gene, such as, without limitation, β-gus,β-actin or 18S rRNA to determine the relative abundance of a given cDNAproduct of a given RNA in a RNA population. Products and reagents forthe fluorescent 5′ nuclease assay are readily available commercially,for instance from Applied Biosystems.

Equipment and software also are readily available for controlling andmonitoring amplicon accumulation in PCR and QRT-PCR according to thefluorescent 5′ nuclease assay and other QPCR/QRT-PCR procedures,including the Smart Cycler, commercially available from Cepheid ofSunnyvale, Calif., the ABI Prism 7700 Sequence Detection System(TaqMan), commercially available from Applied Biosystems. Acartridge-based sample preparation prototype system (GenXpert) combinesa thermal cycler and fluorescent detection device having thecapabilities of the Smart Cycler product with fluid circuits andprocessing elements capable of automatically extracting specific nucleicacids from a tissue sample and performing QPCR or QRT-PCR on the nucleicacid. The system uses disposable cartridges that can be configured andpre-loaded with a broad variety of reagents. Such a system can beconfigured to disrupt tissue and extract total RNA or mRNA from thesample. The reverse transcriptase reaction components can be addedautomatically to the RNA and the QPCR reaction components can be addedautomatically upon completion of the reverse transcriptase reaction.

Further, the PCR reaction may be monitored of production (or loss) of aparticular fluorochrome from the reaction. When the fluorochrome levelsreach (or fall to) a desired level, the automated system willautomatically alter the PCR conditions. In one non-limiting example,this is particularly useful in the multiplexed embodiment describedabove, where a more-abundant (control) target species is amplified bythe first, lower Tm, primer set at a lower temperature than the lessabundant species amplified by the second, higher Tm, primer set. In thefirst stage of the PCR amplification, the annealing step of the cyclingreaction is conducted at a temperature that permits amplification of themore abundant target species. The annealing temperature then isautomatically raised to essentially stop amplification of the moreabundant target species.

In the above-described reactions, the amounts of certain reversetranscriptase and the PCR reaction components typically are atypical inorder to take advantage of the faster ramp times of some thermalcyclers. Specifically, the primer concentrations are very high. Typicalgene-specific primer concentrations for reverse transcriptase reactionsare less than about 20 nM. To achieve a rapid reverse transcriptasereaction on the order of one to two minutes, the reverse transcriptaseprimer concentration was raised to greater than 20 nM, preferably atleast about 50 nM, and typically about 100 nM. Standard PCR primerconcentrations range from 100 nM to 300 nM. Higher concentrations may beused in standard PCR reactions to compensate for Tm variations. However,for purposes herein, the referenced primer concentrations are forcircumstances where no Tm compensation is needed. Proportionately higherconcentrations of primers may be empirically determined and used if Tmcompensation is necessary or desired. To achieve rapid PCR reactions,the PCR primer concentrations typically are greater than 200 nM,preferably greater than about 500 nM and typically about 800 nM.Typically, the ratio of reverse transcriptase primer to PCR primer isabout 1 to 8 or more. The increase in primer concentrations permittedPCR experiments of 40 cycles to be conducted in less than 20 minutes, asdescribed below in Example 2.

A sensitive reverse transcriptase may be preferred in certaincircumstances where either low amounts of RNA are present or a targetRNA is a low abundance RNA. By the term “sensitive reversetranscriptase,” it is meant a reverse transcriptase capable of producingsuitable PCR templates from low copy number transcripts for use as PCRtemplates. The sensitivity of the sensitive reverse transcriptase mayderive from the physical nature of the enzyme, or from specific reactionconditions of the reverse transcriptase reaction mixture that producesthe enhanced sensitivity. One example of a sensitive reversetranscriptase is SensiScript RT reverse transcriptase, commerciallyavailable from Qiagen, Inc. of Valencia Calif. This reversetranscriptase is optimized for the production of cDNA from RNA samplesof <50 ng, but also has the ability to produce PCR templates from lowcopy number transcripts. In practice, in the assays described herein,adequate results were obtained for samples of up to, and even in excessof, about 400 ng RNA. Other sensitive reverse transcriptases havingsubstantially similar ability to reverse transcribe low copy numbertranscripts would be equivalent sensitive reverse transcriptase for thepurposes described herein. Notwithstanding the above, the ability of thesensitive reverse transcriptase to produce cDNA from low quantities ofRNA is secondary to the ability of the enzyme, or enzyme reaction systemto produce PCR templates from low copy number sequences.

As discussed above, the procedures described herein also may be used inmultiplex QRT-PCR processes. In its broadest sense, a multiplex PCRprocess involves production of two or more amplicons in the samereaction vessel. Multiplex amplicons may be analyzed by gelelectrophoresis and detection of the amplicons by one of a variety ofmethods, such as, without limitation ethidium bromide staining, Southernblotting and hybridization to probes, or by incorporating fluorescent orradioactive moieties into the amplicons and subsequently viewing theproduct on a gel. However, real-time monitoring of the production of twoor more amplicons is preferred. The fluorescent 5′ nuclease assay is themost common monitoring method. Equipment is now available (for example,the above-described Smart Cycler and TaqMan products) that permits thereal-time monitoring of accumulation of two or more fluorescentreporters in the same tube. For multiplex monitoring of the fluorescent5′ nuclease assay, oligomers are provided corresponding to each ampliconspecies to be detected. The oligomer probe for each amplicon species hasa fluorescent reporter with a different peak emission wavelength thanthe oligomer probe(s) for each other amplicons species. The accumulationof each unquenched fluorescent reporter can be monitored to determinethe relative amounts of the target sequence corresponding to eachamplicon.

In traditional multiplex QPCR and QRT-PCR procedures, the selection ofPCR primer sets having similar annealing and elongation kinetics andsimilar sized amplicons are desirable. The design and selection ofappropriate PCR primer sets is a process that is well known to a personskilled in the art. The process for identifying optimal PCR primer sets,and respective ratios thereof (primer limiting, that is, limiting theabundance of the PCR primers for the more abundant RNA species in amultiplex PCR experiment to permit the detection of less abundantspecies) to achieve a balanced multiplex reaction also is known. By“balanced,” it is meant that certain amplicon(s) do not out-compete theother amplicon(s) for resources, such as dNTPs or enzyme. Equalizationof the Tm (melting temperature) for all PCR primer sets also isencouraged. See, for instance, ABI PRISM 7700 Sequence Detection SystemUser Bulletin #5, “Multiplex PCR with TaqMan VIC Probes”, AppliedBiosystems (1998/2001).

Despite the above, for very low copy number transcripts, it is difficultto design accurate multiplex PCR experiments, even by limiting the PCRprimer sets for the more abundant control species. One solution to thisproblem is to run the PCR reaction for the low abundance RNA in aseparate tube for the PCR reaction for the more abundant species.However, that strategy does not take advantage of the benefits ofrunning a multiplex PCR experiment. A two-tube process has severaldrawbacks, including cost, the addition of more room for experimentalerror and the increased chance of sample contamination, which iscritical in PCR assays.

A method is therefore provided for performing a multiplex PCR process,including QRT-PCR and QPCR, capable of detecting low copy number nucleicacid species along with one or more higher copy number species. Thedifference between low copy number and high copy number nucleic acidspecies is relative, but is referred to herein as a difference in theprevalence of a low (lower) copy number species and a high (higher) copynumber species of at least about 30-fold, but more typically at leastabout 100-fold. For purposes herein, the relative prevalence of twonucleic acid species to be amplified is more salient than the relativeprevalence of the two nucleic acid species in relation to other nucleicacid species in a given nucleic acid sample because other nucleic acidspecies in the nucleic acid sample do not directly compete with thespecies to be amplified for PCR resources.

As used herein, the prevalence of any given nucleic acid species in agiven nucleic acid sample, prior to testing, is unknown. Thus, the“expected” number of copies of a given nucleic acid species in annucleic acid sample often is used herein and is based on historical dataon the prevalence of that species in nucleic acid samples. For any givenpair of nucleic acid species, one would expect, based on previousdeterminations of the relative prevalence of the two species in asample, the prevalence of each species to fall within a range. Bydetermining these ranges one would determine the -fold difference in theexpected number of target sequences for each species.

The multiplex PCR method involves performing a two- (or more) stage PCRamplification, permitting modulation of the relative rate of productionof a first amplicon by a first primer set and a second amplicon by asecond primer set during the respective amplification stages. The PCRamplification of the second amplification stage is conducted underdifferent reaction conditions (“different reaction conditions” include,without limitation, different temperatures for steps in the PCR cycle,such as the annealing step, or differences in the reagents in the PCRreaction mixture, such as differences in primers and/or primerconcentrations) than in the first amplification stage. By this method,PCR amplifications to produce amplicons directed to a lower abundancenucleic acid species are effectively “balanced” with PCR amplificationsto produce amplicons directed to a higher abundance nucleic acidspecies. Separating the reaction into two or more temporal stages may beachieved by omitting the PCR primer set for any amplicons that are notto be produced in the first amplification stage. The omitted PCR primerset may then be added to the PCR reaction mixture at the beginning ofthe second amplification stage. This is best achieved through use ofautomated processes, such as the GenXpert prototype system describedabove. Two or more separate amplification stages may be used to tailorand balance multiplex assays, along with, or to the exclusion oftailoring the concentration of the respective primer sets.

A second method for temporally separating the PCR amplification processinto two or more stages is to select PCR primer sets with variation intheir respective Tm. Two examples of such a method are provided inExamples 3 and 6, below. In one example, primers for a lower copy numbernucleic acid species would have a higher Tm (Tm₁) than primers for ahigher abundance species (Tm₂). In this process, the first stage of PCRamplification is conducted for a predetermined number of cycles at atemperature sufficiently high that there is substantially noamplification of the higher abundance species. After the first stage ofamplification, the annealing and elongation steps of the PCR reactionare conducted at a lower temperature, typically about Tm₂, so that boththe lower abundance and the higher abundance amplimers are amplified. Itshould be noted that Tm, as used herein and unless otherwise noted,refers to “effective Tm,” which is the Tm for any given primer in agiven reaction mix, which depends on factors, including, withoutlimitation, the nucleic acid sequence of the primer and the primerconcentration in the reaction mixture.

It should be noted that PCR amplification is a dynamic process. Withoutlimitation, when using temperature to modulate the respective PCRreactions in a multiplex PCR reaction, the higher temperature annealingstage may be carried out at any temperature typically ranging from justabove the lower Tm to just above the higher Tm, so long as the reactionfavors production of the amplicon by the higher Tm primer set.Similarly, without limitation, the annealing for the lower temperaturereaction typically is at any temperature below the higher Tm of the lowtemperature primer set that will allow sufficient amplificationefficiency by the lower Tm primer set.

In the example provided above, in the higher temperature stage, theamplicon for the low abundance RNA is amplified at a rate faster thanthe amplicon for the higher abundance RNA (and preferably to thesubstantial exclusion of production of the second amplicon), so that,prior to the second amplification stage, where it is desirable thatamplification of all amplicons proceeds in a substantially balancedmanner, the amplicon for the lower abundance RNA is of sufficientabundance that the amplification of the higher abundance RNA does notinterfere with the amplification of the amplicon for the lower abundanceRNA. In the first stage of amplification, when the amplicon for the lowabundance nucleic acid is preferentially amplified, the annealing andelongation steps may be performed above the higher Tm to gainspecificity over efficiency (during the second stage of theamplification, since there is a relatively large number of low abundancenucleic acid amplicons, selectivity no longer is a significant issue,and efficiency of amplicon production is preferred). It, therefore,should be noted that although favorable in many instances, thetemperature variations may not necessarily result in the completeshutdown of one amplification reaction over another.

In another embodiment, a first primer set with a first Tm may target amore-abundant template sequence (for instance, a control templatesequence) and a second primer set with a higher Tm may target aless-abundant template sequence. In this case, the more-abundanttemplate and the less-abundant template may both be amplified in a firststage at a temperature sufficient to allow sufficient amplification withthe lower Tm primer set, typically at or above the Tm of the first,lower Tm, primer set. When a threshold amount of amplicon correspondingto the more abundant template is reached, the annealing and/orelongation temperature of the reaction is raised to effectively shutdown amplification of the more abundant template.

Selection of three or more sets of PCR primer sets having three or moredifferent Tms (for instance, Tm₁>Tm₂>Tm₃) can be used to amplifysequences of varying abundance in a stepwise manner, so long as thedifferences in the Tms are sufficiently large to permit preferentialamplification of desired sequences to the substantial exclusion ofundesired sequences for a desired number of cycles. In one process, thelowest abundance sequences are amplified in a first stage for apredetermined number of cycles. Next, the lowest abundance and thelesser abundance sequences are amplified in a second stage for apredetermined number of cycles. Lastly, all sequences are amplified in athird stage. As with the two-stage reaction described above, theannealing temperature for each stage may vary, depending on the relativeefficiencies of each single amplification reaction of the multiplexreaction. It should be recognized that two or more amplimers may havesubstantially the same Tm, to permit amplification of more than onespecies of similar abundance at any stage of the amplification process.As with the two-stage reaction, the three-stage reaction may alsoproceed stepwise from amplification of the most abundant nucleic acidspecies at the lowest annealing temperature to amplification of theleast abundant species at the highest annealing temperature.

By this sequential amplification method, an additional tool is providedfor the “balancing” of multiplex PCR reactions besides the matching ofTms and using limiting amounts of one or more PCR primer sets. Theexploitation of PCR primer sets with different Tms as a method forsequentially amplifying different amplicons may be preferred in certaincircumstances to the sequential addition of additional primer sets.However, the use of temperature-dependent sequencing of multiplex PCRreactions may be coupled with the sequential physical addition of primersets to a single reaction mixture.

Also provided is an internal positive control that confirms theoperation of a particular amplification reaction for a negative result.The internal positive controls (IPC) are DNA oligonucleotides that havethe same primer sequences as the target gene (CEA or tyrosinase) buthave a different internal probe sequence. Selected sites in the IPCsoptionally may be synthesized with uracil instead of thymine so thatcontamination with the highly concentrated mimic could be controlledusing uracil DNA glycosylase, if required. The IPCs may be added to anyPCR reaction mastermix in amounts that are determined empirically togive Ct values typically greater than the Ct values of the endogenoustarget of the primer set. The PCR assays are then performed according tostandard protocols, and even when there is no endogenous target for theprimer set, the IPC would be amplified, thereby verifying that thefailure to amplify the target endogenous DNA is not a failure of the PCRreagents in the mastermix. In this embodiment, the IPC probe fluorescesdifferently than the probe for the endogenous sequences. A variation ofthis for use in RT-PCR reactions is where the IPC is an RNA and the RNAincludes an RT primer sequence. In this embodiment, the IPC verifiesfunction of both the RT and PCR reactions. Both RNA and DNA IPCs (withdifferent corresponding probes) may also be employed to differentiatedifficulties in the RT and PCR reactions.

The methods described herein are generally applicable to quantitativePCR and RT-PCR methods. Described herein and the attached manuscriptsare methods for the detection of carcinoembryonic antigen (CEA) andprognosis of adenocarcinomas of the esophagus. The methods describedherein are methods that are equally applicable to the identification ofother micrometastases, including occult micrometastases, in a variety ofother tumor types. The rapid protocols described herein may be run inabout 20 minutes. This short time period permits the assay to be runintraoperatively so that a surgeon can decide on a surgical courseduring a single operation, rather than requiring a second operation, orrequiring the surgeon to perform unneeded or overly broad prophylacticprocedures. For instance, in the surgical evaluation of certain cancers,including breast cancer and melanoma, sentinel lymph nodes are removedin a first operation. The sentinel nodes are later evaluated formicrometastases, and, when micrometastases are detected in a patient'ssentinel lymph node, the patient will need a second operation, therebyincreasing the patient's surgical risks and patient discomfortassociated with multiple operations. In the case of lung or esophagealcancer, intraoperative analysis of lymph nodes can be used to determinethe extent of resection required and/or the need for neoadjuvantchemotherapy. With the ability to determine the expression levels ofcertain tumor-specific markers, such as, without limitation CEA, MUC-1,CK-19, tyrosinase and MART-1, in less than 30 minutes with increasedaccuracy, a physician can make an immediate decision on how to proceed.The rapid test also is applicable to needle biopsies taken in aphysician's office. A patient need not wait for days to get the resultsof a biopsy (such as a needle biopsy of a tumor or lymph node), but cannow get more accurate results in a very short time. The methodsdescribed herein are applicable to detect, without limitation, a varietyof expressed RNAs, whether normal or abnormal, DNA rearrangements or thepresence of additional or abnormal nucleic acids, such as viral nucleicacids.

In the commercialization of the above-described methods for multiplexedand non-multiplexed QRT-PCR and/or QPCR, certain kits for detection ofspecific nucleic acids will be particularly useful. One example of sucha kit would include reagents necessary for the one-tube QRT-PCR processdescribed above. In one example, the kit would include theabove-described reagents, including reverse transcriptase, a reversetranscriptase primer, a corresponding PCR primer set, a thermostable DNApolymerase, such as Taq polymerase, and a suitable fluorescent reporter,such as, without limitation, a probe for a fluorescent 5′ nucleaseassay, a molecular beacon probe, a single dye primer or a fluorescentdye specific to double-stranded DNA, such as ethidium bromide. Theprimers may be present in quantities that would yield the highconcentrations described above. Thermostable DNA polymerases arecommonly and commercially available from a variety of manufacturers.Additional materials in the kit may include: suitable reaction tubes orvials, a barrier composition, typically a wax bead, optionally includingmagnesium; reaction mixtures (typically 10×) for the reversetranscriptase and the PCR stages, including necessary buffers andreagents such as dNTPs; nuclease- or RNase-free water; RNase inhibitor;control nucleic acid(s) and/or any additional buffers, compounds,co-factors, ionic constituents, proteins and enzymes, polymers, and thelike that may be used in reverse transcriptase and/or PCR stages ofQRT-PCR reactions.

A second kit is specific to the above-described multiplex PCR procedure.The kit may include, without limitation, a first PCR primer set for alow abundance nucleic acid, having a first Tm and a second PCR primerset for a more abundant nucleic acid, having a second Tm. The relativeTms of the primer sets are selected for their ability to balance amultiplex PCR reaction according to the methods described herein. In akit for QRT-PCR, the kit also may include: any suitable reversetranscriptase, reverse transcriptase primers specific to the nucleicacids to be amplified, a barrier composition, such as a wax bead, athermostable DNA polymerase and/or a suitable fluorescent reporter, suchas, without limitation, a probe for a fluorescent 5′ nuclease assay, amolecular beacon probe, a single dye primer or a fluorescent dyespecific to double-stranded DNA, such as ethidium bromide. The kit mayinclude a sensitive reverse transcriptase for the detection of lowabundance RNAs. As above, additional materials in the kit may include:suitable reaction tubes or vials, a barrier, typically a wax bead,optionally including magnesium; reaction mixtures (typically 10×) forthe reverse transcriptase and the PCR stages, including necessarybuffers and reagents such as dNTPs; nuclease- or RNase-free water; RNaseinhibitor; control nucleic acid(s) and/or any additional buffers,compounds, co-factors, ionic constituents, proteins and enzymes,polymers, and the like that may be used in reverse transcriptase and/orPCR stages of QRT-PCR reactions.

The above-described kits or cartridges also may include reagents andmechanical components suitable for the manual or automated extraction ofnucleic acid from tissue samples. These reagents are known to thoseskilled in the art and typically are a matter of design choice. Forinstance, in an automated process, tissue may be disruptedultrasonically in a suitable lysis solution provided in the kit orcartridge. The resultant lysate solution may then be filtered and RNAmay be bound to RNA-binding magnetic beads also provided in the kit orcartridge. The beads/RNA may be washed, and the RNA eluted prior to thereverse transcriptase reaction. In the case of automated nucleic acidextraction, the choice of reagents and their mode of packaging (forinstance disposable single-use cartridges) typically are dictated by thephysical configuration of the robotics and fluidics of the specificextraction system, such as Cepheid's GenXpert prototype system.

The constituents of the kits may be packaged together or separately, andeach constituent may be presented in one or more tubes or vials, or incartridge form (a modular unit containing one or more reagents for usein an automated device), as is appropriate. The constituents,independently or together, may be packaged in a variety of states,including without limitation, in lyophilized, glassified, aqueous orother forms as is appropriate.

FIG. 1 is a schematic cross-section diagram of a cartridge 10 for use inthe above-described automated methods. Cartridge 10 includescompartments 20 in which any desired reagent may be stored for use.Compartments 20 are separated by walls 25. Cartridge 10 includesmultiple passageways 30 fluidly connected to a common passageway 40. Avalve member 50 is shown within common passageway 40. Valve member 50controls flow of reagent from individual compartments 20 into commonpassageway 40. Common passageway 40 is fluidly connected to a reactionvessel (not shown) into which reagents from compartments 20 aretransferred. The reagents contained within compartments 20 may include,without limitation, reagents for cell lysis, for nucleic acidpurification, for reverse transcription or for PCR reactions. FIG. 1shows one of many possible permutations of a cartridge device useful inautomating molecular purifications and assays. The cartridge andcompartments may have any desired shape and size, as dictated byempirical factors as well as by designer preference. The choice of andconfiguration of fluid connections and valves also is a matter of designchoice, and may vary greatly.

EXAMPLES Example 1

One-Tube QRT-PCR

With the introduction of real-time, fluorescence-based 5′ nuclease PCR(Gibson, U. E., C. A. Heid and P. M. Williams. 1996. A novel method forreal time quantitative RT PCR. Genome Res. 6:995-1001; Heid, C. A., J.Stevens, K. J. Livak and P. M. Williams. 1996. Real time quantitativePCR. Genome Res. 6:986-994) and instruments such as the ABI PRISMT™ 7700(TaqMan®) sequence detector (Applied Biosystems, Foster City, Calif.,USA), quantitative RT-PCR is now a widely accepted method for measuringgene expression levels. Quantitative RT-PCR is a sensitive technique andis particularly useful for the analysis of samples containing limitedamounts of nucleic acids, such as in clinical tissues (Collins, C., J.M. Rommens, D. Kowbel, T. Godrey, M. Tanner, S. I. Hwang, D. Polikoff,G. Nonet et al. 1998. Positional cloning of ZNF217 and NABC1: genesamplified at 20q13.2 and overexpressed in breast carcinoma. Proc. Natl.Acad. Sci. USA 95:8703-8708). When quantitating these small amounts ofRNA and/or very low-abundance mRNA species, obtaining maximumsensitivity from a quantitative RT-PCR is extremely important. Whileconsecutive rounds of nested PCR are often used to obtain maximumsensitivity, this is difficult to achieve and still maintain accuratequantitation. Furthermore, multiple rounds of PCR increase the risk ofcontamination, a serious problem when working at the desired sensitivitylevels. One-tube RT-PCR (RT and PCR in the same tube using the reversePCR primer for the RT) reduces the risk of contamination when using theABI PRISM 7700 because the reaction tubes are never opened.

Theoretically, a one-tube procedure should have the same sensitivity asa two-step approach (separate RT followed by PCR), but in practice thisis not the case (Battaglia, M., P. Pedrazzoli, B. Palermo, A. Lanza, F.Bertolini, N. Gibelli, G. A. Da Prada, A. Zambelli et al. 1998.Epithelial tumour cell detection and the unsolved problems of nestedRT-PCR: a new sensitive one step method without false positive results.Bone Marrow Transplant 22:693-698). It has been found that thesensitivity of one-tube RT-PCR is limited by the relative nonspecificityof the RT step. This nonspecificity arises from the fact that the RT iscarried out at relatively low temperature and without a hot start, thusallowing nonspecific priming by both the desired RT “reverse” primer andalso from the “forward” PCR primer. As the amount of target decreases inthe input RNA sample, priming artifacts from the cold-start RT processcan compete with, and reduce the efficiency of, PCR amplification of thedesired target sequence. Thus, as RNA input decreases in a one-tubeprocedure, nonspecific side reactions eventually out-compete the desiredreaction, and no specific product is generated. In a two-step or nestedRT-PCR procedure, specificity can be achieved with the use of hot-startPCR and a primer set 5′ upstream from the RT primer. However, this isnot the case in a one-tube procedure unless one is willing to open thereaction tube to add new primers (thus making it a one-tube but two-stepprocedure). It has been hypothesized that by using an external RT primerand keeping the RT and PCR primers separated during the RT step, PCRspecificity and therefore sensitivity in a one-tube RT-PCR should bemaintainable. Here, a modified one-tube RT-PCR assay that greatlyincreases sensitivity and can be used for quantitative RT-PCR on the ABIPRISM 7700 is presented.

Standard one-tube reactions were set up for β-glucuronidase (β-gus) mRNAin 50 μL volumes with the following final concentrations: 10 nM β-gus RTprimer (5′-TTTGGTTGT-CTCTGCCGAGT-3′) (SEQ ID NO: 2), 100 nM each β-gusPCR primer (GUS-F, 5′-C-TCATTTGGAATTTTGCCGATT-3′)(SEQ ID NO: 3); GUS-R,(5′-CCGAGTGAAGATCCC-CTTTTTA-3′)(SEQ ID NO: 4), 100 nM β-gusprobe(5′-6-fam-TGAACAGTCACCGACG-AGAGTGCTGG-tamra-3′) (SEQ ID NO: 5), 1×TaqMan reaction buffer (Applied Biosystems), 5.5 mM MgCl₂, 300 μM eachdNTP, 20 U Rnase inhibitor, 62.5 U SUPERSCRIPT II™ reverse transcriptase(Life Technologies, Rockville, Md., USA) and 1.25 U AmpliTaq Gold®(Applied Biosystems).

In the modified procedure, physical separation between the RT reactionmixture and the PCR primers was achieved by the use of AmpliWax® PCR gem50 (Applied Biosystems). First, the β-gus PCR primers were pipetted intothe PCR plate in a final 5.0-μL volume. One PCR gem 50 was placed ineach well, the wells were capped and the plate was centrifuged brieflyto avoid the adherence of reagents to the tube wall above the waxbarrier. The plate was then heated to 80° C. for 2 min and cooled to 4°C. to produce a wax barrier. A 45 μL upper layer was then pipetted intoeach well. This mixture contained the β-gus RT primer, the RNA, Rnaseinhibitor and SUPERSCRIPT II reverse transcriptase. Both layers wereformulated to contain all of the remaining reaction components (buffer,nucleotides, MgCl₂) at the concentrations described above. The presenceof AmpliTaq Gold in the RT layer is inconsequential because this enzymeis inactive until heated to 95° C.

All reactions were carried out on the ABI PRISM 7700 with the followingthermocycler conditions: 48° C. hold for 30 min, 95° C. hold for 12 min,followed by 40 cycles of 95° C. for 20 s and 60° C. for 1 min. The waxlayer remained intact for the RT step at 48° C. but was melted duringthe 12-min, 95° C. AmpliTaq Gold activation step, thus allowing the twolayers to mix before the PCR begins. Data were analyzed with AppliedBiosystems' sequence detection software.

First, the effect of the wax layer on the fluorescence detection in theTaqMan assay was evaluated to determine the extent of fluorescencequenching by the wax. Using randomly primed cDNA from a lungadenocarcinoma cell line (A549), 20 replicates of PCR for β-gus with andwithout the wax layer were performed. The results showed no decrease inthe overall fluorescence (P=0.935) and no change in the cycle thresholdvalue (P=0.55) between the two groups when compared by the independentsamples t-test.

To compare the sensitivity of the one-tube RT-PCR with and without thewax, serial dilutions of spleen total RNA (Clontech Laboratories, PaloAlto, Calif., USA) from 5 ng to 10 pg were used. The results of theRT-PCR without the wax layer showed that the fluorescence (ΔRn) was weakeven at 5 ng RNA input and decreased further by an average factor of 75%every dilution (FIG. 2A). As a result, the 200-pg sample fell below thethreshold for detection. However, with the use of the wax layer, the ΔRnremained essentially the same down to the 40-pg dilution, and only at 10pg was there a 60% drop in the ΔRn (FIG. 2B). Thus, this modifiedprocedure resulted in at least a 20-fold increase in sensitivity. Theefficiency (E) of the RT-PCR (as calculated by the formulaE.times.10^((1/−s))−1, where “s” is the slope of the standard curve fromthe dilutions) (PE Applied Biosystems User Bulletin #2. 1997. Relativequantitation of gene expression. Applied Biosystems, Foster City,Calif.) was also improved by the use of the wax (67% without wax and 77%with wax). A 10 μL aliquot of each reaction was run out on a 10%non-denaturing polyacrylamide gel and stained with ethidium bromide(FIG. 3). With the wax layer, the 81-bp product, corresponding to theexpected size, was visible in all but the 10-pg RNA dilution(demonstrating the extra sensitivity of TaqMan detection versus ethidiumbromide). In the reactions without wax, however, no reactions produced aclean signal at 81 bp. Instead, there was a smear of nonspecificproducts at all RNA concentrations.

The same experiments were performed using Sensiscript RT® (Qiagen,Valencia, Calif., USA). With this enzyme, PCR product was justdetectable down to the 40-pg dilution even without the use of the waxlayer. However, the total fluorescence continued to drop with eachconsecutive dilution. With the addition of the wax, the ΔRn remainedconstant, and detection was easily achieved down to 10 pg. Notably, theefficiency of this one-tube reaction was near 100% (measured using themethods described above). Thus, the sensitivity of the one-tube RT-PCRfor the 5′ fluorogenic assay in the ABI PRISM 7700 is significantlyenhanced by the use of the AmpliWax PCR gem 50. PCR gems were originallydesigned to facilitate hot start PCR, but this is no longer necessarywith new enzymes for automatic hot start. Here, it is shown that thesesame AmpliWax PCR gems are beneficial in the context of a one-stepquantitative RT-PCR. Furthermore, by eliminating the need to open thePCR tubes the occurrence of cDNA or PCR product contamination wasminimazed. Finally, the preservation of PCR specifically facilitates theamplification of the desired product, and as such, is relevant even innon-quantitative end-point assays.

Example 2

Quantitative RT-PCR in Less than Twenty Minutes

The following is a one-tube two-step assay for quantitative reversetranscription followed by polymerase chain reaction (QRT-PCR), which canbe completed in less than twenty minutes using Cepheid's Smart Cycler.Current methods of QRT-PCR for the 5′ fluorogenic assay in the AppliedBiosystem's 7700 require more than two hours. By altering primer andprobe concentrations and utilizing the fast ramping ability of the SmartCycler, the reverse transcriptase reaction time was reduced to 2 minutesand the PCR time was reduced to 16 minutes using a 1 second denaturationand a 6 second extension for 40 cycles.

PCR reactions were designed for β-glucuronidase (β-gus) andcarcinoembryonic antigen (CEA) cDNA respectively in 25 μl volumes withthe following final concentrations: 400 nM each β-gus PCR primer (GUS-F,5′-CTC ATT TGG AAT TTT GCC GAT T-3′ (SEQ ID NO: 3); (GUS-R, 5′-CCG AGTGAA GAT CCC CTT TTT A-3′)(SEQ ID NO: 4) or CEA primer (CEA-F, 5′-AGA CAATCA CAG TCT CTG CGG A-3′)(SEQ ID NO: 6); (CEA-R, 5′-ATC CTT GTC CTC CACGGG TT-3′) (SEQ ID NO: 7), 200 nM β-gus probe(5′-6-fam-TGAACAGTCACCGACGAGAGTGCTGG-tamra-3) (SEQ ID NO: 5) or 200 nMCEA probe (5′-6-fam-CAA GCC CTC CAT CTC CAG CAA CAA CT-tamra-3) (SEQ IDNO: 8), 1× Platinum Taq reaction buffer, 4.5 mM MgCl₂, 300 μM each dNTP,0.06 U/μl Platinum Taq DNA polymerase (GIBCO BRL).

Tests were run using MgCl₂ at 1.5, 2.5, 3.5 and 4.5 mM concentrationsand it was determined that 4.5 mM is optimal for this assay. The cDNAfor these reactions was generated from gene specific reversetranscriptase reactions for GUS and CEA using a 250 ng input of RNA froman A549 cell line (GUS) and fresh lymph node RNA that was positive forCEA.

RT-PCR reactions were designed for β-gus mRNA and CEA mRNA in 25 μlvolumes with the same PCR concentrations as above and the followingreverse transcriptase concentrations: 60 nM β-gus reverse transcriptaseprimer (5′-TTTGGTTGTCTCTGCCGAGT-3′) (SEQ ID NO: 2) or CEA reversetranscriptase primer (5′-GTG AAG GCC ACA GCA T-3′) (SEQ ID NO: 9), 1 μlSensiscript and 0.8 U/μl RNase inhibitor. The RNA input for the RT-PCRwas 400 ng A549 and 25 ng lymph node.

The denaturation step was first optimized by comparing 1, 2, 5, 7 and 10second denaturation at 95° C. in combination with a 30 second extensionfor 40 cycles. Platinum Taq activation was done at 95° C. for 30seconds. The results of this testing show no significant loss ofsensitivity between a 1 second denaturation versus a 10 seconddenaturation for either gene. Next, the extension step was optimized bycomparing 3, 5, 7, 10, 13, 15 and 30 second extension at 64° C. incombination with a 15 second denaturation for 40 cycles. Platinum Taqactivation was done at 95° C. for 30 seconds.

The results of this testing show minimal loss of sensitivity ofapproximately one and a half cycles from 30 second extension to 3 secondextension for GUS. For CEA, no significant loss is seen from 30 secondextension to 3 second extension.

Next, the combined effect of altering denaturation/extension time wasevaluated by comparing a ⅓ second PCR to a 2/15 second PCR over 40cycles. The results show a 2.2 and 1.1 cycle loss in sensitivity for GUSand CEA, respectively. A 2/15 second PCR requires 22 minutes while a ⅓second PCR requires 16 minutes, thus this insignificant loss insensitivity is well worth cutting the reaction time by 6 minutes.

In an attempt to reduce the ramping time from denaturation to extension,the effect of decreasing the denaturation temperature from 95° C. to 90°C. to 85° C. was evaluated. For GUS, there is no significant loss insensitivity when denaturation is done at 95° C. or 85° C. For CEA, thereaction failed when denaturation was done at 85° C. but no significantloss in sensitivity was seen from denaturation at 95° C. to 90° C. Theamount of time gained by doing a 90° C. versus a 95° C. denaturation isabout 1.5 minutes over 40 cycles.

Taq activation time was evaluated and no significant loss in sensitivityfor either gene by decreasing Taq activation from 30 seconds to 10seconds was found.

After optimizing the PCR conditions, the reverse transcriptase reactionwas optimized. The reverse transcriptase reaction was done in a totalvolume of 15 μl. After completion of the reaction, the mixture was heldat 70° C. at which time the PCR components (total volume, 10 μl) wereadded. Reverse transcriptase reaction times of 2, 3, 5, 7 and 10 minuteswere compared. The reverse transcriptase reactions were combined withPCR conditions that included a 1 second denaturation at 95° C. and a 5second extension at 64° C. for 40 cycles for both genes.

The results of these reverse transcriptase reaction time trials show aloss in sensitivity of 1.1 cycle for GUS and 1.81 cycles for CEA from a10 minute reverse transcriptase reaction to a 2 minute reversetranscriptase reaction. Next, the total effect of decreasing the RT-PCRtimes on the sensitivity of the assay was evaluated by comparing thefollowing RT-PCR conditions: 1) a “gold standard” with a 10 minutereverse transcriptase reaction followed by 10 second denaturation and 15second extension for 40 cycles, total run time of 38 minutes, 2) a 5minute reverse transcriptase reaction followed by optimized PCRconditions of 1 second denaturation and 5 second extension for 40cycles, total run time of 20 minutes, 3) a 2 minute reversetranscriptase reaction followed by optimized PCR conditions, total runtime of 17 minutes, and 4) a “quick” RT-PCR with a 2 minute reversetranscriptase reaction followed by 1 second denaturation and 3 secondextension for 40 cycles, total run time of 15 minutes. For Gus, the“gold standard” RT-PCR had a Ct (number of cycles required to reach apredetermined threshold, reference fluorescence level) of 25.88±0.78while the 2 min reverse transcriptase reaction with optimized PCRconditions had a Ct of 29.42±0.7 showing a total cycle difference of3.54. For CEA, the “gold standard” RT-PCR had a Ct of 29.94±2.2 whilethe 2 min RT with optimized PCR conditions had a Ct of 34.92±0.5 showinga total cycle difference of 4.98.

In an attempt to increase the sensitivity of the shorter protocol, theeffects of increasing the primer concentrations for Gus and CEA wereevaluated. The experiment described above was repeated with an increaseof the concentration of the RT primer from 60 nM to 100 nM and the F/RPCR primers from 400 nM to 800 nM. The results of this test show a 2.3cycle difference (4.98 with low primer concentrations) between the goldstandard versus the 2 min optimal protocol for CEA and a 1.63 (3.54 withlow primer concentrations) cycle difference for Gus. This small loss insensitivity is insignificant considering that the total RT-PCR time wasreduced from 38 minutes to 17 minutes.

The PCR efficiency for CEA was evaluated using the optimized conditionsof 1 second denaturation and 6 second extension for 40 cycles on adilution series of the fresh lymph node cDNA. The correlationcoefficient of this assay is 0.9974 indicating excellentreproducibility. The PCR efficiency can be calculated as follows:E=10^((1/−S))−1,

where S equals the slope of a standard curve of a serial dilution oftemplate, for which Ct is plotted versus the log DNA concentration.Therefore, the PCR efficiency for this assay is 96.7%.

Next, the RT-PCR efficiency of the assay was evaluated using a 2 minreverse transcriptase reaction followed by 40 cycles of PCR using theoptimized conditions. A fresh lymph node RNA 2.times.dilution series wasprepared in 400 ng spleen RNA. RT-PCR for both CEA and GUS wereperformed. The mean GUS Ct was 28.39±1.36. The efficiency for this assaywas greater than 100 percent. The same assay was done using an equalmixture of Superscript II and Sensiscript rather than only Sensiscript.The efficiency of this assay was closer to 100 percent.

Example 3

Rapid QRT-PCR: Multiplexed Assay

The Smart Cycler is currently capable of 4-color fluorescence detectionand therefore allows multiplexing of QRT-PCR reactions. One goal is tomultiplex internal controls for RT-PCR, an endogenous reference genecontrol to correct for RNA input, and the target gene (for example CEA)all in one tube. Initial tests multiplexing βGUS and CEA worked well atmoderate CEA mRNA levels but failed when very low levels of CEA werepresent. Thus, the sensitivity of this reaction was not adequate formicrometastasis detection. One method to overcome this is to limit theamount of PCR primer used for the endogenous control gene. Theoreticallythis allows the rare CEA mRNA species to more effectively compete forPCR reagents, especially in later cycles. Attempts to do this withβ-Gus, or a second endogenous control gene (18s ribosomal RNA), alsofailed to give adequate sensitivity.

It was hypothesized that the problem lay in the initial cycles, whencompetition between the two PCR reactions was most critical. To overcomethis, but still carry out the assay in a single tube with no extrahandling, the 18S rRNA (and βGUS) endogenous control primers wereredesigned so that the annealing temperature was 10° C. below that ofthe CEA primers (all primers used in the QRT-PCR reactions describedherein are listed below in Table 1). PCR was then carried out in two20-cycle steps, the first with an anneal/extend temperature of 64° C.and the second with an anneal-extend temperature of 53° C. The multiplexreaction reagent concentrations were the same as those used in thesingleplex reaction with the following modifications. The target geneprimer concentrations were at 400 nM while that of the endogenouscontrols' were at 100 nM, the reverse transcriptase primerconcentrations were at 60 nM each and the cycling conditions weremodified as previously mentioned. Theoretically the 18S rRNA primerswould not function in the first 20 cycles and the CEA amplificationcould proceed without competition. In the next 20 cycles, CEA PCRproduct would already be boosted to the point that it could competeefficiently with the 18S rRNA PCR. The results of this experiment areshown in FIG. 4. Panels A and C show the results for 18S rRNA and CEArespectively when run in a singleplex using optimal conditions. Panels Band D show 18S rRNA and CEA multiplexed. Note that while these reactionswere multiplexed in the same tube, with different fluorescent dyes, thesoftware does not allow two dyes to be plotted on the same graph. In thesingleplex reaction, 18SrRNA crossed the 30-fluorescence unit thresholdat 10 cycles (Panel A). Using the new PCR primers, and modifiedprotocol, the 18S rRNA PCR reaction did not cross threshold until 26cycles (Panel B), 6 cycles after the anneal/extend temperature wasdropped to 53° C. This reaction was expected to cross threshold at 30cycles (20+10), thus it appears that there is some 18S rRNA PCRamplification occurring during the first 20 cycles, even at 64° C. Inthe CEA reactions the singleplex reached threshold at 33.5 cycles (PanelC) while the multiplexed CEA reached threshold at 34.5 cycles. Thus onlyone cycle sensitivity was lost in this reaction. TABLE 1 Name SequenceCEA probe CAA GCC CTC CAT CTC CAG CAA CAA CT (SEQ ID NO: 8) CEA RT GTGAAG GCC ACA GCA T (SEQ ID NO: 9) CEA-F77 AGA CAA TCA CAG TCT CTG CGG A(SEQ ID NO: 6) CEA-R77 ATC CTT GTC CTC CAC GGG TT (SEQ ID NO: 7) 18Sprobe TGC TGG CAC CAG ACT TGC CCT C (SEQ ID NO: 10) 18S-F89 CCC TGT AATTGG AAT GAG TCC AC (SEQ ID NO: 11) 18S-R89 GCT GGA ATT ACC GCG GCT (SEQID NO: 12) 18S-F2- CCC TGT AAT TGG AAT GAG T low Temp (SEQ ID NO: 13)18S-R2- GCT GGA ATT ACC GCG low Temp (SEQ ID NO: 14) Gus probe TGA ACAGTC ACC GAC GAG AGT GCT GG (SEQ ID NO: 5) GUS RT TGG TTG TCT CTG CCG A(SEQ ID NO: 15) Gus 81F CTC ATT TGG AAT TTT GCC GAT T (SEQ ID NO: 3) Gus81R CCG AGT GAA GAT CCC CTT TTT A (SEQ ID NO: 4) Gus 80F- CTC ATT TGGAAT TTT GCC low Temp (SEQ ID NO: 16) Gus 80R- CG AGT GAA GAT CCC CTT lowTemp (SEQ ID NO: 17)

Of note, the above-described CEA primers were designed to span thejunction between exons 6 and 7 of the CEA mRNA. The primers also amplifysequences spanning the junction between exons 2-3 of the CEA mRNA. Thisprimer set was selected to yield superior selectivity to certain earlierdescribed primer sets. The addition of one or more flanking nucleotidesof the CEA sequence (FIG. 5, GenBank Accession No. XM_(—)012777) to the5′ or 3′ ends of either of the CEA primers would not appreciably affectthe above-described assays, except with respect to expected changes inthe Tm of the primer set. Other CEA-specific primers may be selectedfrom the same general regions (exon 2-3 and exon 6-7 junctions) that theabove-described CEA forward and reverse primers are selected to the sameor similar effect as the above-described CEA primers. Preferably, anyselected primer sets will yield an amplicon of less than about 100bases, which adds to the ability to conduct a rapid QRT-PCR assay.

Example 4

Prognostic Value of Quantitative Reverse Transcription-Polymerase ChainReaction in Node-Negative Esophageal Cancer Patients

Introduction

The incidence of adenocarcinoma of the esophagus is increasing at analarming rate, exceeding that of any other solid tumor. Up to 50% ofpatients present with advanced disease, yielding an overall 5-yearsurvival of 10-13%. As with other tumor types, survival of esophagealcancer patients is strongly predicted by histological evidence of lymphnode involvement. Although current histological methods for lymph nodestaging provide reliable information about populations of patients, theycannot predict individual patient outcome within that population. Forexample, 30-50% of histologically node-negative esophageal cancerpatients will suffer disease recurrence within 5 years, despite apotentially curative resection. There is a circumstantial body ofevidence indicating that this primary treatment failure is attributableto micrometastatic spread of the tumor that went undetected by routinehistological evaluation. Thus, there is a clear need for more sensitivedetection of lymph node micrometastases, thereby allowing moreindividualized prognosis and treatment planning of esophageal cancerpatients.

The main problems with current lymph node evaluation are sampling errorand poor sensitivity for detecting individual tumor cells or small tumorfoci. Histological examination only samples a very small percentage ofeach lymph node, and it has been calculated that a pathologist has onlya 1% chance of detecting a micrometastatic focus of three tumor celldiameter. Immunohistochemical staining for tumor markers improves thesensitivity of micrometastasis detection and, when combined with serialsectioning to reduce the sampling error, results in upstaging of asignificant number of patients. This technique has been used inesophagus cancer, and 17% of histologically negative nodes were found tocontain micrometastatic disease. In this report, IHC3-positive lymphnodes correlated with disease recurrence, but these findings werequestioned in a later study in which IHC did not show any prognosticvalue. Other studies have used molecular methods such as RT-PCR todetect micrometastases. RT-PCR is capable of detecting the mRNA fortumor markers, such as CEA, cytokeratin 19, cytokeratin 20, and others,in a variety of tissues, including blood, bone marrow, and lymph nodes,that are histologically cancer free. In esophageal cancer, RT-PCR hasbeen used on several small series of patients, but the clinicalsignificance of RT-PCR-positive nodes is not known because littleclinical follow-up has been reported. In other tumor types, studies haveshown that RT-PCR improves sensitivity, but poor specificity andfalse-positive results in control lymph nodes from patients withoutcancer have made the clinical application of this information difficult.False positives are, at least in part, attributable to the previouslydescribed phenomenon of ectopic gene expression, which results in verylow background levels of expression of most genes in all tissue types.Thus, although previous studies have used qualitative, gel-based RT-PCRmethods, it is now becoming apparent that this simple plus/minus methodfor detection of tumor markers is not always a reliable sign ofmicrometastases. With the introduction of the fluorescent 5′ nucleaseassay, QRT-PCR is now a relatively simple technique. It was thereforehypothesized that a quantitative analysis would offer significantbenefits over gel-based RT-PCR and would allow accurate prediction ofdisease recurrence in histologically node-negative esophageal cancerpatients. The objectives of the present were 3-fold: (a) to determinedthe ability of QRT-PCR to accurately distinguish between background geneexpression of CEA in lymph nodes and clinically relevant levels that arediagnostic of true micrometastasis; (b) to use real-time QRT-PCR toanalyze lymph nodes from 30 node-negative esophageal cancer patients andcorrelated the results with disease recurrence; and (c) to compareQRT-PCR with standard gel-based RT-PCR on the same samples. It was foundthat QRT-PCR can easily discriminate background expression from truemetastatic disease, QRT-PCR is both sensitive and specific forpredicting disease recurrence in nodenegative esophageal cancerpatients, and QRT-PCR has greater specificity than gel-based RT-PCR.From these results, it is believed that quantitation addresses all ofthe problems that have kept RT-PCR from becoming a useful clinical testfor micrometastatic disease.

Materials and Methods

Patients: Tissue from 140 paraffin blocks containing 387 lymph nodeswere studied from 30 patients who underwent curative resection forhistologically lymph node-negative esophageal cancer. The Section ofThoracic Surgery performed all surgeries at the University of PittsburghMedical Center between 1991 and 1998. Vital status and recurrenceinformation was obtained from a combination of medical record review,personal contact with the Primary Care Physician, and deathcertificates. Follow-up data were confirmed for all patients as ofAugust 2001. The median follow-up time for surviving patients was 49months (range, 28-91 months). Demographics and clinical characteristicswere collected (Table 2). Tissue blocks from 10 primary tumors (8adenocarcinoma and 2 squamous cell carcinoma) and 4 histologicallypositive lymph nodes were obtained as positive controls. Negativecontrol (benign) lymph nodes were obtained from patients who underwentesophageal surgery for causes unrelated to cancer (hernia repairs andantireflux procedures) and whose lymph nodes were removed incidentally.TABLE 2 Clinical Characteristics of the study population QRT-PCR ResultPatients Negative Positive Characteristic (n = 30) (n = 19) (n = 11)Gender Male 22 15 7 Female 8 4 4 Months of follow up Median 36.0 44.628.0 Range  5-90.6  5-90.6 6.3-57.4 Mean Age at Diagnosis 68.3 68.8 67.5Adjuvant Therapy Chemotherapy 16 7 9 Radiation 9 3 6 Lymphadenopathy onscan 8 3 5 Tumor Type Adenocarcinoma 26 18 8 Squamous cell 4 1 3 pTcategory* pT1 10 8 2 pT2 5 4 1 pT3 10 5 5 Stage* I 10 8 2 IIA 15 9 6Mean Number of Nodes 12.5 12 15 examined (range) (2-31) (2-31) (3-23)*Four patients who received chemotherapy had no tumor at time of surgery

Tissue and RNA Isolation: All tissues used in the study wereformalin-fixed, paraffin-embedded archival specimens obtained from thePathology tissue banks. H&E-stained slides were also retrieved for eachtissue block and were examined to confirm the original node-negativediagnosis. Tissue blocks were mounted on a microtome, and 5-15 5.0 cMsections were cut and placed in 2.0 ml of RNase-free tubes. At the sametime, 2 5.0 μM sections were cut (first and last cuts) and mounted onmicroscope slides for immunohistochemical staining with antibodiesagainst CEA. RNA was isolated using methods described previously(Godfrey, T. E., Kim, S.-H., Chavira, M., Ruff, D. W., Warren, R. S.,Gray, J. W., and Jensen, R. H. Quantitative mRNA expression analysisfrom formalin-fixed, paraffin-embedded tissues using 5′ nucleasequantitative RT-PCR. J. Mol. Diagn., 2: 84-91, 2000) and stored in RNAsecure resuspension solution (Ambion, Austin, Tex.). The RNA was DNasetreated with the DNA free kit (Ambion) and quantitatedspectrophotometrically.

QRT-PCR: QRT-PCR was carried out using the 5′ nuclease assay and anApplied Biosystems 7700 Sequence Detection Instrument (TaqMan). CEAexpression was measured relative to the endogenous control gene, β-gus,using the comparative CT method described previously (Godfrey, T. E.,Kim, S.-H., Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W., andJensen, R. H. Quantitative mRNA expression analysis from formalin-fixed,paraffin-embedded tissues using 5′ nuclease quantitative RT-PCR. J. Mol.Diagn., 2: 84-91, 2000; Tassone, F., Hagerman, R. J., Taylor, A. K.,Gane, L. W., Godfrey, T. E., and Hagerman, P. J. Elevated Levels of FMR1mRNA in carrier males: a new mechanism of involvement in the fragile-Xsyndrome. Am. J. Hum. Genet., 66: 6-15, 2000). All QRT-PCR assays werecarried out at two RNA inputs, 400 and 100 ng, and duplicate reactionswere set up for each concentration. Thus, the reported CEA expressionlevels are an average of four independent QRT-PCR reactions. RT-negativecontrols were run for all samples using 400 ng of RNA but omitting thereverse transcriptase. Template-negative controls were also run on eachPCR plate. A calibrator RNA sample was amplified in parallel on allplates to allow comparison of samples run at different times (Godfrey,T. E., Kim, S.-H., Chavira, M., Ruff, D. W., Warren, R. S., Gray, J. W.,and Jensen, R. H. Quantitative mRNA expression analysis fromformalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitativeRT-PCR. J. Mol. Diagn., 2: 84-91, 2000; PE Applied Biosystems userbulletin #2. Relative quantitation of gene expression, Perkin-Elmer,Corp., Norwalk, Conn., 1997.).

For maximum sensitivity and to eliminate the risk of crosscontamination, a single-tube QRT-PCR procedure described previously wasused (Raja, S., Luketich, J. D., Ruff, D. W., Kelly, L. A., and Godfrey,T. E. A Method for increased sensitivity of one-step quantitativeRT-PCR. Biotechniques, 29: 702-705, 2000). In this procedure, physicalseparation of the reverse transcription reaction mixture (RT) and thePCR primers using a wax layer results in a more specific and sensitiveRT-PCR. The PCR primers were pipetted into the PCR plate in a 10 μlvolume. One Ampliwax PCR gem 50 (Applied Biosystems) was then placed ineach well, and the plate was heated to 80° C. for 2 min and cooled to 4°C. to produce the wax barrier. A 40 μl upper layer was then pipettedinto each well. The final concentrations of the reaction components wereas follows: 1×PCR buffer A, 300 nM each deoxynucleotide triphosphate,3.5 mM MgCl₂, 0.4 unit/μl RNase Inhibitor, 1.25 units/μl Superscript IIreverse transcriptase (Life Technologies, Inc., Gaithersburg, Md.), 0.06unit/μl Amplitaq Gold (Applied Biosystems), 20 nM reverse transcriptaseprimer, 200 nM of each PCR primer, 200 nM probe (β-Gus primers and probewere at 100 nM), and 100 or 400 ng total RNA. The oligonucleotidesequences used are shown in Table 3. All RT-PCR reactions were carriedout on the ABI 7700 with the following thermocycler conditions: 48° C.hold for 40 min, 95° C. hold for 12 min followed by 45 cycles of 95° C.for 15 s, and 64° C. (60° C. for β-gus) for 1 min. Data were analyzedusing Sequence Detection Software (Applied Biosystems) with thresholdsset at 0.03 for CEA and 0.045 for β-gus. TABLE 3 Oligonucleotidesequences used for CEA and-Gus RT-PCR Oligo- nucleotide B-Gus CEAForward CTCATTTGGAATTTTGCCGATT AGACAATCACAGTCTCTGCGG primer (SEQ ID NO:3) (SEQ ID NO: 6) Reverse CCGAGTGAAGATCCCCTTTTTA ATCCTTGTCCTCCACGGGTTprimer (SEQ ID NO: 4) (SEQ ID NO: 7) RT primer TGGTTGTCTCTGCCGAGTGAAGGCCACAGCAT (SEQ ID NO: 15) (SEQ ID NO: 9) Probe*TGAACAGTCACCGACGAGAGTGCTGG CAAGCCCTCCATCTCCAGCAACAACT (SEQ ID NO: 5)(SEQ ID NO: 8)*TaqMan probes were labeled with 5′ 6-carboxyfluorescein and 3′6-carboxytetramethylrhodamine.

Gel-based RT-PCR Analysis: To avoid the possibility of PCR productcontamination, all PCR plates from QRT-PCR runs were stored unopeneduntil the quantitative analyses were complete. PCR products from the two400 ng RNA input reactions and the 400 ng No-RT control were thenseparated on a 4% agarose gel, stained with ethidium bromide, andvisualized on a gel documentation system. Patients were classified asRT-PCR positive if a correctly sized band was observed in both of theduplicate reactions but not in the No-RT control.

Statistical Analysis: Comparisons of CEA levels in control tissuessamples (FIG. 6) were conducted with the Mann-Whitney U Test. Forpathologically negative lymph nodes from esophageal cancer patients, theprimary end point was disease recurrence measured from the time ofsurgery to the time of diagnosed recurrence or last date of follow-up.The highest CEA levels from each patient's tissue blocks were used toconstruct a ROC curve using recurrence as the gold standard. The CEAexpression level cutoff value was identified that produced the mostaccurate classification, and that level was used to classify patients asQRT-PCR positive or negative for risk of recurrence. Because a secondset of patients was not available for prospective validation of thecutoff, the cutoff selection procedure was evaluated statistically bycross validation, and the SDs of ROC curve statistics were calculated bybootstrap resampling (Davison, A. C., and Hinkley, D. V. BootstrapMethods and Their Applications. Cambridge, United Kingdom: CambridgeUniversity Press, 1997). Kaplan-Meier disease-free and overall survivalcurves were plotted for clinical and pathological factors includingstandard RT-PCR and QRT-PCR results. The analysis of disease-freesurvival was conducted by log-rank tests, and multivariate analysis wasperformed by constructing Cox proportionate hazards models. The list ofpredictors included categorical factors gender, tumor pathology,classification by RT-PCR and QRT-PCR, preoperative chemotherapy and/orradiotherapy, and pathological T stage, as well as quantitative factorsof CEA expression level, age, and number of nodes removed forexamination. All comparisons between models were based on differencesbetween likelihood ratio tests for nested models. The adequacy of theproportional assumption was checked two ways: by plotting the logcumulative hazard by log time and by plotting Schoenfeld residuals andestimating the correlation between the regression coefficient and time.

RESULTS—Characteristics of Node-negative Patients: TNM classification ofthe 30 patients with node-negative esophageal cancer includedT₁N₀M₀(n=10), T₂N₀M₀ (n=5), and T₃N₀M₀ (n=10). One patient with biopsydiagnosed cancer had no evidence of cancer on the resection specimen,and four patients who received neoadjuvant therapy had no detectabletumor remaining at the time of resection. The location of the primarytumor included 25 lower third, 5 mid, and 0 upper esophageal sites.There were 26 adeno and 4 squamous cell cancers. There were 22 males and8 females, and the median age of the patients at diagnosis was 70 years.Seventeen of the 30 patients have died, 10 from their cancers and 7 fromother causes. For surviving patients, the median follow-up was 49 months(range, 28-91 months). The median overall survival was 36 months. A fullbreakdown of patient characteristics is provided in Table 4, below.

Quantitative Analysis of CEA Expression: Initially, RNA was isolated andanalyzed from three sources, distinct from the node-negative studygroup, and included primary esophageal tumors, lymph nodes that werehistologically positive for metastases (N₁), and benign lymph nodes frompatients without cancer. CEA expression was detected in all tumors, andN₁ nodes and in 50% of benign lymph nodes (FIG. 6). Individual, pairwisecomparisons were carried out using a Mann-Whitney U test, and expressionlevels in both tumor and N₁ nodes were found to be significantly higherthan in benign lymph nodes (P=0.002 and 0.0021, respectively). CEAexpression was slightly higher in tumor samples than in N₁ lymph nodes,but this was not statistically significant (P=0.171).

Analysis of histologically negative (N₀) lymph nodes from the studygroup demonstrated a wide range of CEA expression. Expression rangedfrom undetectable to one node with CEA expression equal to that seen inhistologically positive lymph nodes. Data from the most highlyexpressing node from each patient, in conjunction with diseaserecurrence information, were analyzed using a ROC curve analysis. Fromthis analysis, an expression level cutoff that most accurately predictedrecurrence was determined (FIG. 7). The area under the ROC curve was0.88 with a 95% confidence of 0.71 to 0.97, indicating thatclassification accuracy is significantly better than chance alone. Ofthe 140 tissue blocks analyzed, 12 (9%) had CEA expression levels abovethe cutoff point, and a total of 11 patients (34%) had one or moreblocks with expression above the cutoff. These 11 patients wereclassified as having QRT-PCR evidence of occult micrometastases.Sections from all but 1 (patient 28) of the “most positive” tissueblocks were negative for immunohistochemical staining with antibodiesagainst CEA.

Gel-based Analysis of CEA Expression: A total of 25 (18%) tissue blockswere positive for CEA expression using the gel-based assay, and 15 (50%)patients had at least one positive block in this analysis. In 2 of the10 lymph nodes from patients with benign disorders, a CEA PCR productwas present on the gel-based assays. A comparison of the quantitativeand gelbased assays showed that all samples positive on gels also gavesignals in the TaqMan assay.

Occult Metastases and Recurrence: Of the 30 patients studied, 10suffered disease recurrence and died by the end of the study. Of theremaining 20 patients, 7 died from other causes and 13 remain alive withno evidence of recurrent disease. One patient suffered an earlyanastomatic recurrence that was treated with photodynamic therapy andradiotherapy. This patient was later diagnosed with, and died of, smallcell lung cancer. Using the most accurate cutoff for the quantitativeassay, a total of 11 patients were classified as QRT-PCR positive, and 9of these suffered recurrence. The same 9 patients were identified asRT-PCR positive using the gel-based assay, along with 6 other patientswho did not recur. Sensitivity and specificity for predicting diseaserecurrence using the QRT-PCR assay was 90 and 90%, with a positivepredictive value of 82%. Using the gel-based assay, the sensitivity andspecificity were 90 and 70% with a positive predictive value of 60%.FIG. 8 shows disease-free survival for the patient cohort classified byRT-PCR (A) and QRT-PCR(C) as well as overall survival (B and D).Disease-free survival of patients who were RT-PCR negative was 94% whenusing either assay. Patients with a positive classification had poorerprognosis by either method. The log-rank test indicates that bothQRT-PCR(P=0001) and gel-based RT-PCR(P=0038) are significant predictorsof recurrence in node-negative esophageal cancer patients. Overallsurvival was also worse in QRT-PCR- or RT-PCR-positive patients(P=0.0006 and 0.106, respectively).

Besides QRT-PCR and RT-PCR, the only other clinical, pathological, ortreatment factors found to predict disease recurrence in this cohortwere pathological T stage (log-rank test, P=0.0777) and preoperativechemotherapy and/or radiotherapy (log rank test, P=0.0307). In amultivariate analysis, the likelihood ratio statistics from Coxregression models showed that the classification of CEA as a binaryvariable, QRT-PCR positive or negative, was a significant andindependent predictor of recurrence when compared with pathological Tstage and preoperative chemotherapy or radiotherapy.

Discussion

Lymph node involvement is the strongest prognostic factor in many solidtumors, and detection of lymph node micrometastases has received muchinterest in the recent literature. Current lymph node evaluationinvolves microscopic examination of H&E-stained tissue sections andsuffers from two major limitations: (a) single tumor cells, or smallfoci of cells, are easily missed; and (b) only one or two tissuesections are studied, and thus the vast majority of each node is leftunexamined. Serial sectioning can overcome the issue of sampling error,and IHC can identify individual tumor cells. The combination of thesemethods, however, is too costly and time consuming for routine analysisand is limited to special cases such as sentinel lymph node examination.RT-PCR overcomes the problem of sampling error because larger amounts oftissue can be analyzed, and several reports indicate that RT-PCRidentifies more positive lymph nodes than IHC. This was also the case inthe present study, where only one of the QRT-PCR-positive lymph nodeblocks was positive by IHC analysis.

Recent studies also show that non-QRT-PCR positivity correlates withdisease recurrence, but the specificity reported in these studies islow. In one study by Liefers et al. (Liefers, G. J., Cleton-Jansen, A.M., van de Velde, C. J., Hermans, J., van Krieken, J. H., Cornelisse, C.J., and Tollenaar, R. A. Micrometastases and survival in stage IIcolorectal cancer [see comments]. N. Engl. J. Med., 339: 223-228,1998.), 14 of 26 histologically N₀ colon cancer patients had evidence ofmicrometastatic disease using RT-PCR, and the remaining 12 were RT-PCRnegative. Of the 12 RT-PCR N₀ patients, only 1 recurred during the6-year follow-up period, whereas of the 14 RT-PCR N₁ patients, 7suffered recurrence. Thus, using recurrence as an end point, this studyachieved a sensitivity of 88%. The specificity, however, was only 61%,because 7 patients with RT-PCR-positive nodes did not recur. Otherstudies have shown a similar low specificity for non-QRT-PCR in melanomapatients. Shivers et al. (Shivers, S. C., Wang, X., Li, W., Joseph, E.,Messina, J., Glass, L. F., DeConti, R., Cruse, C. W., Berman, C.,Fenske, N. A., Lyman, G. H., and Reintgen, D. S. Molecular staging ofmalignant melanoma: correlation with clinical outcome. JAMA, 280:1410-1415, 1998) achieved 86% sensitivity and 51% specificity, andBostick et al. (Bostick, P. J., Morton, D. L., Turner, R. R., Huynh, K.T., Wang, H. J., Elashoff, R., Essner, R., and Hoon, D. S. Prognosticsignificance of occult metastases detected by sentinel lymphadenectomyand reverse transcriptase-polymerase chain reaction in early-stagemelanoma patients. J. Clin. Oncol., 17: 3238-3244, 1999) reported 100%sensitivity and 67% specificity. This low specificity, along with aninability to accurately control for inter-run variability, has limitedthe potential of RT-PCR in a clinical setting.

In the current study, the use of TaqMan RT-PCR to quantitatively assayCEA expression and detect occult micrometastases in lymph nodes ofhistologically N₀ esophageal cancer patients was evaluated. Thisquantitative analysis resulted in the definition of a clinicallyrelevant CEA expression level cutoff and the overcoming of the problemsassociated with background, or ectopic, CEA expression reported byothers. Using this approach, 11 of 30 patients were identified as beingQRT-PCR positive, and 9 of these patients suffered disease recurrence.Only 1 of the 19 patients who were QRT-PCR negative suffered recurrenceduring the course of this study. Interestingly, of the 20 patients whodid not recur, 11 had detectable CEA expression higher than that seen incontrol lymph nodes (higher than background levels), and two were abovethe cutoff level for predicting disease recurrence. In some cases, thiscould indicate the presence of limited nodal disease that was cured bysurgery, because even pN₁ patients can have an expected 5-year survivalof˜20%. CEA expression in the remaining samples could possibly be aresult of either individual disseminated tumor cells that are unable tosurvive and are possibly undergoing apoptosis, cell-free RNA in thelymph system as a result of tumor cell apoptosis at the primary site, orcontaminating cells introduced inadvertently by the surgeon.

Both disease-free survival and overall survival were significantlyhigher in the QRT-PCR-negative patients compared with QRT-PCR-positivepatients. Furthermore, disease-free survival in the QRT-PCR-positivegroup was only 27% at 3 years, indicating that micrometastatic diseasemay be as clinically significant as histological N₁ disease. With theexception of one patient, only one positive tissue block was identifiedper patient, indicating limited disease spread. All positive lymph nodeswere locoregional and would therefore confer N₁ status, rather thanM_(1a), as defined by celiac or cervical lymph node involvement. Thelimited nodal involvement in these histologically N₀ patients emphasizesthe need for adequate lymph node sampling, from different nodalstations, during staging procedures.

Although the subset of patients with node-negative disease has beenrelatively small in the past, the dramatic increase in esophageal cancerhas led to surveillance programs that are identifying patients withearly stage disease more frequently. Methods for accurate staging ofthese patients will thus become even more important. Comparison hereinof the quantitative data with gel-based analysis of the same samplesshowed that QRT-PCR improves the test specificity while maintaining thesame high sensitivity. QRT-PCR also gives objective results, which areamenable to automated, hands-free analysis, with minimal risk of PCRproduct cross-contamination. Such automation will be essential ifQRT-PCR is to become a routine clinical assay.

In addition, the quantitative procedure allows for use of rigorouscontrols to confirm the accuracy and reliability of the assay from runto run. For example, in the described experiments, a calibrator samplewas run on all PCR plates to correct for day to day variability. Usingthis methodology, reproducibility tests indicate that the 95% confidencelimits on measurements are 0.511 cycles (data not shown). The ability toaccurately assess the reproducibility of the quantitative assay will beessential if RT-PCR is to be used in a clinical setting. In a gel-basedassay, this level of assay verification is not attainable. It isacknowledged that the predictive ability of classifying patients bytheir CEA levels determined by QRT-PCR will be optimistic because thecutoff value was evaluated in the same patients in whom it wasdetermined. It is likely that if the same procedure for the mostaccurate cutoff were applied to a second set of patients, theclassification would be less successful. To this end, a reanalysis ofthe sensitivity, specificity, and classification accuracy was conductedby cross-validation (n=10000). The cross-validation estimates ofspecificity were 0.82 and accuracy of 0.82 (compared with 0.90 and 0.90,respectively, in the original sample). Thus, the classification successof the original sample was slightly overstated, but even aftercorrection for this optimism, classification by QRT-PCR remains animprovement over the gel-based assay.

In conclusion, these data demonstrate that QRT-PCR can detect, with highspecificity, micrometastatic disease in histologically negative lymphnodes of esophagus cancer patients. It also has been shown that thepresence of micrometastatic disease is a strong, independent predictorof cancer recurrence and that quantitation is superior to standardRT-PCR assays. Quantitative RT-PCR should be able to identify whichpatients with early stage esophageal cancer are at high risk forrecurrence and who might benefit from additional therapy. Finally, thisquantitative approach should result in similar benefits in other tumortypes. TABLE 4 Survival and RT-PCR data of individual patients OutcomeSurvival CEA status Patient T Adjuvant therapy Vital Disease DiseaseQRT- RT- ID stage Chemotherapy Radiation Status Status^(a) Overall FreePCR PCR  1 2 None None Alive NED 43.9 43.9 − −  2 1 None None Alive NED28.4 28.4 − −  3^(b) 1 Post Post Alive NED 30.8 30.8 − −  4 1 Pre NoneDead Other 17.8 17.8 − −  5 1 None None Alive NED 90.6 90.6 − −  6 3 PreNone Alive NED 60.6 60.6 − +  7 1 None None Alive NED 50.5 50.5 − +  8 2None None Alive NED 75.6 75.6 − −  9 1 None None Dead Other 38.1 38.1 −− 10 3 Pre None Alive NED 69.1 69.1 − + 11 2 None None Alive NED 44.644.6 − − 12 3 Pre Pre Dead Other 31.7 31.7 − − 13 1 None None Dead Other63.1 63.1 − − 14^(b) 0^(c) Pre Pre Alive NED 35.0 35.0 + + 15^(b) 1 NoneNone Dead Other 31.8 31.8 + + 16 0^(d) None None Alive NED 49.4 49.4 − −17 2 None None Alive NED 36.9 36.9 − − 18 1 None None Alive NED 65.265.2 − − 19 0^(c) Pre None Dead Other 56.3 15.8 − + 20 3 Pre Pre DeadOther 32.2 32.2 − − 21 3 None None Dead NED 5.0 5.0 − − 22 3 Pre NoneDead Recurred 24.8 24.3 + + 23 1 Pre None Dead Recurred 57.4 53.0 + + 243 Pre None Dead Recurred 28.0 7.9 + + 25^(b) 3 None None Dead Recurred*6.3 5.0 + + 26 3 Pre Pre Dead Recurred 11.1 10.6 + + 27 2 Pre Pre DeadRecurred 14.6 10.4 + + 28 0^(c) Pre Pre Dead Recurred 46.4 8.3 + + 29 3Pre Pre Dead Recurred 26.3 18.7 + + 30 0^(c) Pre Pre Dead Recurred 31.927.0 + +^(a)NED, no evidence of disease.^(b)Patients with squamous cell carcinoma.^(c)Patients who had no residual tumor after chemotherapy.^(d)Patient with cancer on biopsy, but not at resection.

Example 5

Intra-Operative Quantitative RT-PCR Detects Nodal Metastasis in Patientswith Esophageal Cancer

Introduction

Surgical decisions in esophageal cancer and other malignancies are oftenbased on intra-operative frozen section analysis of lymph nodes. The5-year survival of patients with esophageal cancer remains poor at 5-10%due to the presence of advanced disease in many patients at the time ofinitial presentation. If local and regional lymph nodes arehistologically negative however, there is a dramatic improvement in the5-year survival. Nevertheless, 30-50% of histologically node negativepatients suffer disease recurrence. This is most likely due to thelimitations of current staging techniques in the detection ofmicrometastases. As a result, other techniques such asimmunohistochemistry or reverse transcriptase-polymerase chain reaction(RT-PCR) have been used in attempts to detect histologically occultmicrometastases.

Studies on esophagus, colon, melanoma and breast cancers have shown thatRT-PCR can detect histologically occult micrometastases, and may predictrecurrence. These data have been criticized however, due to theoccurrence of false positive results in control samples and thesubsequent low specificity and positive predictive value of the RT-PCRassay. It has been shown that Quantitative RT-PCR (QRT-PCR) allows thedistinction of background, ectopic, gene expression from truemicrometases and can therefore avoid false positives and increasespecificity for predicting disease recurrence. The next goal is to beable to provide the surgeon with this critical information at a timewhen important surgical decisions are made, intra-operatively.

Described in this Example is the development and testing of an extremelyrapid QRT-PCR assay that can be carried out in less than 30 minutes. Tovalidate the rapid assay, it is compared to standard QRT-PCR and toclinical outcomes in patients with esophageal cancer.

Materials and Methods

Patients

For the retrospective analysis, lymph nodes were studied from 30patients (Table 6) who underwent curative resection for histologicallylymph node-negative esophageal cancer. All surgeries were performed inthe Division of Thoracic Surgery at the University of Pittsburgh MedicalCenter between 1991 and 1998. Clinical follow up was obtained from themedical records and was confirmed for all patients as of August 2001.The median clinical follow up time for all patients was 36 months andfor the surviving patients was 49 months (range 28-91 months). TABLE 6Clinical Characteristics of the study population. QRT-PCR ResultPatients Negative Positive Characteristic (n = 30) (n = 17) (n = 13)Gender Male 22 14 8 Female 8 3 5 Months follow up Median 36 43.9 31.8Range 5-90.5 5-90.5 6.3-75.6 Mean Age at Diagnosis 68.3 68.4 68.2 TumorType Adenocarcinoma 26 16 10 Squamous cell 4 1 3 Stage* 0\ 1 1 0 I 10 82 IIA 15 8 7 Median Number of Nodes 12.5 12 15 Examined*4 patients who received chemotherapy had no tumor at time of surgery.\1 patient had cancer on biopsy but not at resection.

Prospective analysis, lymph node sample were obtained from 12 patientsundergoing either staging or resection for esophageal cancer identifiedby the Section of Thoracic Surgery at the University of PittsburghMedical Center from 1999 to 2000 (Table 7). Control nodes from patientswithout cancer were obtained incidentally from patients undergoingabdominal surgery for anti-reflux procedures. At the time of excision,one half of each lymph node was frozen in liquid nitrogen for analysisand the remainder was sent for routine pathological analysis. Theexercise tissue was part of the routine clinical course at ourinstitutional and, as such, no additional tissue was removed for anypurely research purpose. All tissues were collected as part of anongoing, TRB approved, Esophagus Cancer Risk Registry protocol at theUniversity of Pittsburgh. TABLE 7 Tumor stage and histology for patientsin the rapid QRT-PCR study on fresh frozen lymph nodes. Patient stageTumor Type 12 IIA adeno 13 IIA adeno 14 I adeno 15 IIA adeno 16 IIAadeno 17 IIA squamous 18 III adeno 19 III adeno 20 III adeno 21 IIIadeno 22 III adeno 23 III adenoTissue and RNA Isolation

All tissues used in the retrospective study were foramalin-fixed,paraffin-embedded archival specimens obtained from the Pathology tissuebanks. Hematoxylin and eosin stained slides were also retrieved for eachtissue block and were examined to confirm the original node-negativediagnosis. Tissue blocks were mounted on a microtome and 5-15, 5.0 μMsections were cut and placed in 2.0 ml RNase free tubes. At the sametime, 2, 5.0 μM sections were cut (first and last sections) and mountedon microscope slides for H&E staining as well as immunohistochemicalstaining with antibodies against CEA. RNA was isolated using previouslydescribed methods (Godfrey T E, Kim S-H, Chavira M, Ruff D W, Warren RS, Gray J W et al. Quantitative mRNA expression analysis fromformalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitativeRT-PCR. J. of Molecular Diagnostics 2000; 2(2):84-91) and quantitatedspectrophotometrically.

The fresh frozen lymph node tissues were embedded in OCT compound and10-15, 4.0 μM sections were cut. At the same time, 2, 4.0 μM sectionswere cut (first and last sections) and mounted on microscope slides forH&E staining. The remaining sections were placed in 1.5 ml RNase freetubes with the lysis buffer from the RNeasy Mini kit (Qiagen, Valencia,Calif.). RNA was extracted using the manufacturers recommended protocolwith the following modifications. Centrifugation times over 1 minutewere reduced to 1 minute, and the RNA was reconstituted in 60 μl RNAsecure resuspension solution (Ambion, Austin, Tex.). Although most ofthe samples were processed together, five samples were processedindividually to determine the median extraction time per sample. The RNAyield and purity was determined spectrophotometrically for qualitycontrol purposes.

Quantitative Reverse Transcription-PCR—QRT-PCR was carried out using the5′ nuclease assay. Rapid QRT-PCR was carried out on the Cepheid SmartCycler® (CSC) real-time DNA amplification and detection system asdescribed below. Standard QRT-PCR was carried out on the AppliedBiosystems 7700 Sequence Detection Instrument (TaqMan®) using a one-tubeQRT-PCR procedure described herein.

Rapid QRT-PCR—CEA expression was measured relative to the endogenouscontrol gene, β-glucuronidase (β-GUS) using the comparative C_(T) methoddescribed previously (Godfrey T E, Kim S-H, Chavira M, Ruff D W, WarrenR S, Gray J W et al. Quantitative mRNA expression analysis fromformalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitativeRT-PCR. J. Molecular Diagnostics 2000; 2(2):84-91; Tassone F, Hagerman RJ, Taylor A K, Gane L W, Godfrey T E, Hagerman P J. Elevated levels ofFMR1 mRNA in carrier males: a new mechanism of involvement in thefragile X syndrome. Am. J. Hum. Genet. 2000; 66(1):6-15.). All QRT-PCRassays were carried out on 400 ng of total RNA in triplicate. Both CEAand β-GUS PCR primers were designed and tested not to amplify genomicDNA. However, control reactions were still run using RNA without reversetranscription (NO-RT control) and water (no-template control) in placeof cDNA as PCR template. Together these control reactions rule out thepossibility that signal is generated from either genomic DNAcontamination of the RNA, or PCR product contamination of the reagents.In addition, a calibrator RNA sample was amplified in parallel on allruns to allow comparison of samples run at different times (Godfrey etal. (2000); PE Applied Biosystems User Bulletin #2. ABI Prism 7700Sequence Detection System: Relative Quantitaion of Gene expression.1997. Norwalk, Conn., Perkin Elmer Corp.), and to determinereproducibility of the assay.

The final concentrations of the reaction components were as follows:1×PCR Platinum Taq buffer, 300 nM each dNTP, 4.5 mM MgCl₂, 0.8 U/μlRNase Inhibitor, 1.25 μl Sensiscript reverse transcriptase (Qiagen,Valencia, Calif.), 0.06 U/μl Platinum Taq (Gibco, Gaithersburg, Md.), 60nM RT primer, 400 nM each PCR primer, 200 nM probe and 400 ng of totalRNA. The total RNA input for the fresh tissue analysis was 5 μl of thesample. For the fixed tissue analysis, 5 μl of an 80 ng/μl dilution ofthe RNA stock was used. In the rapid assay, the RT reaction was carriedout without the PCR primers or probe, then the tube was opened and theprimers and probe were added. This was necessary because a standardone-step QRT-PCR lacked the sensitivity to detect rare messages. Theoligonucleotide sequences used were as follows: β-GUS RT primer 5′ TGGTTG TCT CTG CCG A 3′ (SEQ ID NO: 15), β-GUS forward PCR primer 5′ CTCATT TGG AAT TTT GCC GAT T 3′ (SEQ ID NO: 3), β-GUS reverse PCR primer 5′CCG AGT GAA GAT CCC CTT TTT A 3′ (SEQ ID NO: 4), β-GUS probe 5′-Vic TGAACA GTC ACC GAC GAG AGT GCT GG 3′ (SEQ ID NO: 5), CEA RT primer 5′ GTGAAG GCC ACA GCA T 3′ (SEQ ID NO: 9), CEA forward PCR primer 5′ AGA CAATCA CAG TCT CTG CGG A 3′ (SEQ ID NO: 6), CEA reverse PCR primer 5′ ATCCTT GTC CTC CAC GGG TT 3′ (SEQ ID NO: 7) and CEA probe 5′-Fam CAA GCCCTC CAT CTC CAG CAA CAA CT 3′ (SEQ ID NO: 8). Rapid QRT-PCR reactionswere carried out on the CSC with the following thermocycler conditions;48° C. hold for 5 minutes, 70° C. for 60 seconds (for the addition ofthe PCR primers and probe), 95° C. hold for 30 seconds (for Platinum Taqactivation) followed by 45 cycles of 95° C. for 2 seconds and 64° C. for15 seconds. Data was analyzed with SmartCycler Software (Ver 1.0) usingthe second derivative method for determining the threshold. For thefixed tissue analysis, a 30 minute RT reaction was required because ofthe reduced sensitivity associated with the degradation inherent infixed tissue RNA samples.

Statistical Analysis—The predictive validity of rapid QRT-PCRdetermination of CEA expression level was evaluated by proportionatehazards regression for disease-free survival. Disease-free survival wasdefined as the time from surgery to the time of diagnosed recurrence ofesophageal cancer. Deaths due to other causes and patients alive withoutdisease as of Aug. 1, 2001 were censored. CEA expression level was alsoused to classify patients as either QRT-PCR positive or negative. Thecutoff level for classification, determined by ROC curve analysis(DeLong E R, DeLong D M, Clarke-Pearson D L. Comparing the areas undertwo or more correlated receiver operating characteristic curves: anonparametric approach. Biometrics 1988; 44(3):837-845; Heagerty P J,Lumley T, Pepe M S. Time-dependent ROC curves for censored survival dataand a diagnostic marker. Biometrics 2000; 56(2):337-344), was defined asthe CEA expression level value that produced the most accurateclassification using disease recurrence as the standard. Sensitivity andspecificity of QRT-PCR results were calculated for diagnosing occultmetastasis. Patients classified as QRT-PCR positive or negative based onthe classification method were tested for differences in disease-freesurvival with the log rank test.

Results

Archived Tissue Analysis

Initially, archived, histologically negative (N₀) lymph nodes from 30patients with esophageal cancer were analyzed (FIG. 9). Of the 30 tissueblocks analyzed, 13 had CEA expression higher than the ROC curvedetermined cutoff (CEA expression greater than 0.183). In this group,nine patients suffered disease recurrence and died by the end of thestudy, 2 patients have died from other causes and 2 are alive withoutdisease. Of the remaining 17 QRT-PCR negative patients, 1 suffereddisease recurrence at 5 months and died by the end of this study, 5 havedied from other causes and 11 remain alive and disease free (Table 8).Using the rapid QRT-PCR assay, the sensitivity and specificity forpredicting disease recurrence were 90% and 80% respectively and 83% ofpatients in the cohort were classified correctly. FIG. 10 shows theKaplan-Meier disease-free survival curves for QRT-PCR positive andnegative patients. Survival of patients who were QRT-PCR negative andpositive was 94% and 20% respectively at 5 years. The survival functionsfor these two groups were significantly different (p=0.001, log ranktest). TABLE 8 Rapid QRT-PCR results on 30 pN₀ esophagus cancer patientsusing a CEA expression cut-off of 0.183. Sensitivity was 90% (9/10) andspecificity was 80%. Recurrence (true +ve) No recurrence (true −ve)QRT-PCR positive 9 4 QRT-PCR negative 1 16

Using the proportionate hazards model for disease free survival, therelative risk (RR) of an increase of one unit of CEA expression was 1.52(1.11-2.09, p-0.027) and that of having CEA expression greater than0.183 was 3.78 (1.34-10.7, p=0.0007) compared to CEA expression lessthan or equal to 0.183.

Fresh Tissue Analysis

Fresh frozen sections of lymph nodes from patients undergoing minimallyinvasive staging for esophageal cancer, or benign esophageal disorders,were analyzed. In the 11 lymph nodes from the benign cases, rapidQRT-PCR detected very low levels of CEA expression in only 2 lymph nodes(patients 8 and 11, FIG. 11). This low level in the benign casespresumably represented background, ectopic CEA expression. The highestlevel of CEA expression in any benign node was 34-fold lower than thelowest expressing positive node (FIG. 11), which had only a small focusof tumor involvement.

Of the 26 lymph nodes from 12 patients with esophageal cancer, 12 (6patients) were ultimately shown to be positive on final pathology. In 2(2 patients) of these lymph nodes however, intra-operative frozensection analysis was negative and N₁ status was determinedpost-operatively. Rapid QRT-PCR showed CEA expression levels in these 2nodes to be in the range of the histologically positive nodes (patient18 and 19, FIG. 11). In the 6 patients who had histologically nodenegative esophageal cancer, only one had a single node with increasedCEA expression by rapid QRT-PCR (patient 15, FIG. 11). This patient hada clinical recurrence of his esophageal cancer at a follow-up of 16.5months.

Analysis of the calibrator samples on each run revealed that, over 13separate runs, the standard deviation of the delta Ct value was 0.14cycles (˜10% standard deviation of relative expression measurements).Additionally, the QRT-PCR required approximately twenty-five minutes forall 40-cycle runs. With an RNA isolation time of 7 minutes, the entireassay takes around 30 minutes. Therefore, this is a very rapid andreproducible assay.

Discussion

In many malignancies, as in esophagus cancer, TNM staging yields thebest prognostic information. The absence of nodal involvement isassociated with a more localized lesion suggesting that a cure may beobtained by surgical resection alone. Nodal metastases however, areindicators of tumor spread beyond the site of the primary tumor and, assuch, necessitate systemic treatment. Although the role of chemotherapyin esophageal cancer remains controversial, some trials have showntrends indicating the benefit of pre-operative chemotherapy. Oneadvantage of this approach is that more patients are able to completethe chemotherapy protocol, than would otherwise do so if they were toreceive treatment following a very morbid surgical procedure. Ongoingprospective, randomized clinical trials should resolve the utility ofpre-operative chemotherapy in esophagus cancer patients in the nearfuture. With the advent of minimally invasive surgical approaches tostaging, there exists the potential to stage prior to resection andoffer pre-operative chemotherapy when appropriate. Therefore, theaccurate determination of nodal stage prior to attempted curativeresection could become very important in the decision-making process forpatients with esophageal cancer.

Currently, intra-operative nodal staging is performed by frozensectioning and H&E staining. While this methodology has at least a 93%correlation with the gold standard of formalin fixed, H&E stained tissueexamination, both methods suffer significant limitations. Specifically,30-50% of histologically node negative esophagus cancer patients sufferdisease recurrence despite potentially curative resection. This findingis most likely due to sampling error that is inherent in theconventional methodologies, as well as a lack of sensitivity fordetecting micrometastases. Single tumor cells, or small foci of cells,are easily missed since only one or two 4 μM sections are studied andthe vast majority of each node is left unexamined. Serial sectioning oflymph nodes can reduce the sampling error and thus minimize thislimitation. For example, in studies on breast cancer, serial sectioningof lymph nodes, combined with immunohistochemistry, led to an upstagingin an additional 10-23% of nodes that were negative by routinehistopathologic evaluation. This method is not routinely used howeverdue to the significant time and labor involved in serial sectioning ofnumerous lymph nodes from each case.

In the last decade, many studies have used RT-PCR to detect tumorrelated mRNAs in lymph nodes in attempts to improve the sensitivity ofmicrometastasis detection. Studies on several cancer types have reportedthat RT-PCR is a reasonable prognostic indicator, but despite this,RT-PCR has not made its way into clinical practice. The main reasons forthis are poor specificity (40-60%), false positive results in controlnodes from patients without cancer and the lack of standardized assaysfor multi-center trials. In other work, it has been shown that aquantitative approach to RT-PCR (QRT-PCR) can overcome false positivesdue to background or ectopic gene expression, and can increase thespecificity for predicting disease recurrence Godfrey T E, Raja S,Finkelstein S D, Kelly L A, Gooding W, Luketich J D. Quantitative RT-PCRPredicts Disease Recurrence in lymph Node-Negative Esophagus CancerPatients. Proceedings from the 2001 Annual Meeting of the AmericanAssociation of Cancer Researchers 42. Mar. 1, 2001. Due to thetechniques used in that study, however, the RNA isolation and RT-PCRassay requires 4-6 hours to complete. As a result, that method can onlybe utilized post-operatively. Intraoperative QRT-PCR can be a realityonly if it is performed rapidly enough to yield a result in a time framecomparable to intra-operative frozen section analysis (approximately20-30 minutes). Along with rapid RNA isolation and reverse transcriptionprotocols, this requires extremely fast PCR ramping times such as thoseattainable with the Roche Light Cycler® or the Cepheid Smart Cycler®.The Cepheid instrument was used herein due to the relative ease of use(no glass capillaries), independent control of each reaction site,availability of four color multiplexing and the potential for automatedsample preparation and QRT-PCR. With this instrument it has been shownthat a QRT-PCR assay can be performed on OCT embedded tissue sectionswithin 30 minutes from RNA extraction to result. With modifications tothe protocol reported here, QRT-PCR can be carried out in 18.5 minutes(data not shown). Using our rapid assay, a retrospective analysis of RNAfrom archival tissues with a median patient follow-up of 36 months wasperformed. Data showed that the rapid assay predicted disease recurrencewith a sensitivity and specificity of 90% and 80% respectively. Thisdata was comparable with the slower, and more traditional, TaqMan basedanalysis (90% and 90%). In the fresh tissue analysis, it was possible todistinguish all the histologically positive nodes from the benigncontrol nodes in under 30 minutes. Using the expression level of CEA, itwas also possible to characterize pathologically negative nodes ashaving an expression pattern that resembled either benign orpathologically positive nodes.

From this data, it appears that rapid QRT-PCR is at least as sensitiveas fixed tissue examination and is more sensitive than intraoperativefrozen section examination. This is demonstrated by the 2 positive nodesthat were deferred or misdiagnosed during the intraoperativeexamination. To confirm this data, study of a much larger number offresh lymph nodes is planned with the object to correlate the resultswith pathologic staging. As the data set matures, QRT-PCR will becorrelated with with recurrence. This will permit determination of anoptimal CEA expression cut-off value in the fresh tissue data set sincethis may be different from the cut-off for archived tissues. Due to thepreliminary fresh tissue data, and problems inherent in analysis ofarchived tissues, it is possible that specificity and positivepredictive values will improve with analysis of fresh tissues.

Further development of this technology will be necessary to bringintraoperative QRT-PCR from bench to bedside. In a clinical setting, theQRT-PCR assay has to be a simple and automated process with minimalhandling. Towards this end, the goal is to develop a cartridge basedprocessor for automated analysis. It is envisioned that the end productwill be a single-use, disposable cartridge capable of both RNA isolationand quantitative RT-PCR set up. When integrated with a rapid cycling,real-time quantitative thermal cycler, this system will provide aQRT-PCR result from frozen sections in less than 25 minutes. Upon itsdevelopment, and the identification of new and accurate markers, thistechnology could also be used for molecular analysis of surgical marginsas well as an adjunct to fine needle aspirate cytology. Thus, molecularinformation regarding micrometastases and completeness of resectioncould, for the first time, be made available intraoperatively to thesurgeon. Furthermore, the availability of an automated and reproducibleQRT-PCR format will allow the true molecular diagnostic value of QRT-PCRto be evaluated in standardized multi-center trials.

Example 6

Description of a Novel Method for Multiplexing Polymerase ChainReaction.

Introduction

Despite the enormous power and flexibility of the Polymerase ChainReaction (PCR), this technology has been slow to find its way intoclinical diagnostic laboratories. In large part, this is due to the factthat the PCR process (from sample processing to reaction set-up and dataanalysis) is labor intensive, prone to contamination and technicallyquite demanding. As a result, false positive and false negative resultsare frequent and constitute a major concern in the clinical setting.Even so, most major clinical diagnostic laboratories are still running ahandful of internally validated, PCR-based assays for a variety ofapplications such as viral detection and minimal residual diseasedetection in leukemia patients. Technological advances that simplifyand, if possible, automate PCR procedures would greatly enhance thereproducibility and reliability of these assays.

One such technological advance, the introduction of rapid cycling,quantitative PCR instruments, is opening up new potential uses formolecular testing. The ability to complete PCR assays in less than 30minutes now makes it possible to carry out time dependent, point of caremolecular diagnostics such as testing for group B streptococcus inpregnant women and nosocomial agents in immunocompromised patients. Incancer diagnostics, such rapid tests bring the possibility of primarycancer diagnosis on core biopsies or fine needle aspirates (FNA) whilethe patient is still in the clinic, tumor profiling at the time ofsurgery to determine response to chemotherapy and intraoperative testingof lymph nodes and surgical margins to determine extent of disease andtreatment options. For example, Reverse Transcription-PCR (RT-PCR) hasbeen shown in many studies to improve the sensitivity of cancer celldetection in lymph nodes of cancer patients otherwise staged as nodenegative. These patients are at higher risk for disease recurrence andmay benefit from more aggressive therapy. In the case of lung cancer forexample, patients with mediastinal lymph node involvement may benefitfrom neoadjuvant chemotherapy but lymph node status needs to bedetermined prior to major surgical intervention. This can be achievedthrough minimally invasive surgery or even ultrasound guided FNA.Accurate intraoperative lymph node diagnosis would allow negativepatients to undergo complete tumor resection while surgery would behalted in node positive patients to allow administration ofchemotherapy. A similar scenario exists in patients with breast canceror melanoma. In both diseases, sentinel lymph node positivity results ina complete lymph node dissection.

Since current intraoperative lymph node analysis methods are not verysensitive (˜70% in breast cancer (Weiser M R, Montgomery L L, Susnik B,Tan L K, Borgen P I, Cody H S. Is routine intraoperative frozen-sectionexamination of sentinel lymph nodes in breast cancer worthwhile? AnnSurg Oncol. 2000;7:651-5) and 47% in melanoma (Fitzgerald R C,Triadafilopoulos G. Recent developments in the molecularcharacterization of Barrett's esophagus. Dig. Dis. 1998;16:63-80.)) manypatients are not diagnosed as lymph node positive until after completionof the surgery. These patients then have to undergo a second surgicalprocedure to complete the lymph node dissection. A more sensitive,intraoperative lymph node assessment would clearly benefit thesepatients. This example illustrates just one potential use of rapidPCR-based assays but many more are likely to be forthcoming in the nearfuture. Once again however, advances are needed in PCR technology tomake these exciting possibilities practical in the clinical diagnosticlaboratory.

The recent introduction of fluorescence-based PCR, and RT-PCR, hasgreatly simplified data analysis steps by eliminating the need forpost-PCR processing and gel electrophoresis. Added benefits of thistechnology include reduced assay contamination (since PCR tubes arenever opened) and, of course, the ability to obtain highly accurate andreproducible quantitative results. Quantitation not only enhances theclinical utility of many potential diagnostic tests but also providesthe ability to verify assay sensitivity and reproducibility from test totest with the use of external quality control standards. Internalquality controls are also required, including amplification ofendogenous control sequences to check for quality and quantity of thetemplate DNA or RNA, and internal positive controls to verify that theassay worked in the case of a negative result. While these ‘internal’controls can actually be set up in separate tubes, it would beadvantageous if all assays could be run (multiplexed) in the same PCRtube, taking advantage of different colored fluorogenic probes todistinguish the PCR products. Unfortunately, standard multiplex PCR doesnot work well when the amplification targets are not present in similarabundance at the beginning of the reaction and it is especiallydifficult to maintain accurate quantitation over a wide range of targetconcentrations. The dynamic range of a quantitative multiplex can beimproved to some degree by severely limiting the PCR primerconcentration for the more abundant gene in the PCR reaction. While thisapproach works reasonably well for standard RTPCR assays, the concept ofprimer limiting goes against the requirements of a rapid PCR assay,where higher primer concentrations are necessary to maintainamplification. Here, a method is described for multiplex PCR that hasthe same sensitivity and quantitative dynamic range as a singleplex PCR,and that can also be performed in a rapid RT-PCR reaction. It isdemonstrated herein that this methodology is applicable to multipledifferent amplification targets and that, with the addition of asynthetic oligonucleotide mimic, it provides for an internallycontrolled, rapid, multiplex QRT-PCR. The value of this method in cancerdiagnostics is demonstrated here by the detection of tumor cells inlymph nodes of esophageal cancer and melanoma patients. This rapid,intraoperative, QRT-PCR may eventually be used for more accurateintraoperative cancer staging and will therefore result in moreappropriate and timely treatment of cancer patients.

Materials and Methods

Tissues and RNA isolation: All patient tissue samples were obtainedthrough University of Pittsburgh IRB approved protocols. The lymph nodesfrom patients with esophagus cancer, and the benign nodes (obtainedincidentally from patients undergoing anti-reflux procedures) weresnap-frozen in liquid nitrogen and RNA was extracted using the RNeasyMini kit (Qiagen, Valencia Calif.) following the manufacturer'sprotocol. The histologically positive and negative lymph nodes frommelanoma patients were obtained as formalin-fixed and paraffin embeddedarchived samples and RNA was extracted using previously describedprotocols (Godfrey T E, Kim S H, Chavira M, Ruff D W, Warren R S, Gray JW, Jensen R H. Quantitative mRNA expression analysis fromformalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitativereverse transcription-polymerase chain reaction. J Mol Diagn.2000;2:84-91.).

Development of the Rapid PCR Assay: Rapid PCR was initially tested usingcDNA synthesized from human colon total RNA (Ambion, Austin Tex.) astemplate. PCR primers and probes for β-glucuronidase (β-gus) andcarcinoembryonic antigen (CEA) were designed to be cDNA specific withamplicon sizes of 81 and 77 base pairs, respectively. Known pseudogenesfor CEA were avoided and control PCR on 200 ng of genomic DNA wasnegative. PCR was performed in 25 μL reactions containing 800 nmol/L ofeach primer, 200 nmol/L of each probe (primer and probe sequences areshown in Table 9) and the PCR master mix [1× Platinum Taq reactionbuffer, 4.5 mmol/L MgCl₂, 300 μmol/L of each dNTP and 0.09 U/μL PlatinumTaq DNA polymerase (Invitrogen, Carlsbad Calif.)]. Assays were performedon the Cepheid Smartcycler™ (Cepheid, Sunnyvale, Calif.) and analyzedwith Smartcycler software v1.2b. PCR denaturation (95° C.) times of 10,7, 5, 2 and 1 second, and annealing/extension (64° C.) times of 30, 15,10, 7, 5 and 3 seconds were initially tested on a single cDNA input. Theoptimal rapid PCR cycling conditions (1 sec denaturation followed by a 6sec annealing/extension) were then used to compare the rapid PCRprotocol with more conventional cycling times of 10 sec denaturation and30 sec annealing/extension. This was carried out on a serially dilutedCEA standard curve to determine any effect of the rapid PCR protocolover a wide range of CEA inputs. The CEA cDNA was serially diluted (8×)in a constant background of β-gus template (purified β-gus PCR product)in amounts that were empirically determined to give constant β-gus cyclethreshold (Ct) values between 19 and 20 cycles. Similar experiments werecarried out to optimize and validate the tyrosinase assay. TABLE 9Primer and probe sequences Name Sequence (5′→3′) CEA-FAGACAATCACAGTCTCTGCGGA (SEQ ID NO: 6) CEA-R ATCCTTGTCCTCCACGGGTT (SEQ IDNO: 7) CEA probe-FAM FAM-AGCUGCCCAAGCCCU-BHQ1* (SEQ ID NO: 18) CEA RTGTGAAGGCCACAGCAT (SEQ ID NO: 9) Gus 81F CTCATTTGGAATTTTGCCGATT (SEQ IDNO: 3) Gus 81R CCGAGTGAAGATCCCCTTTTTA (SEQ ID NO: 4) Gus 80FCTCATTTGGAATTTTGCC (SEQ ID NO: 16) Gus 80R CGAGTGAAGATCCGCTT (SEQ ID NO:17) Gus probe Texas Red- TGAACAGTCACCGACGAGAGTGCTGG-BHQ2 (SEQ ID NO: 5)Gus RT TTTGGTTGTCTCTGCCGAGT (SEQ ID NO: 2) Tyrosinase-FACTTACTCAGCCCAGCATCATTC (SEQ ID NO: 19) Tyrosinase-RACTGATGGCTGTTGTACTCCTCG (SEQ ID NO: 20) Tyrosinase FAM- probeTCTCCTCTTGGCAGATTGTCTGTAGCCGA BHQ1 (SEQ ID NO: 21) Tyrosinase RTCGTTCCATTGCATAAG (SEQ ID NO: 22) Internal Alexa 546- ControlAGCATCATCCTCTGCATGGTCAGGTCAT-BHQ1 Probe (SEQ ID NO: 23)*C and U were changed to their propyne pyrimidine counterparts

Development of the rapid reverse transcription assay. The RNA input forthe RT-PCR was a mixture of 400 ng obtained from the A549 lungadenocarcinoma cell line and 25 ng from a lymph node with metastaticesophageal adenocarcinoma. RT-PCR reactions were performed for β-gus andCEA mRNA in 25 μL containing the PCR master mix described above plus 60nmol/L β-gus and CEA RT primers, 1 μL SensiScript reverse transcriptase(Qiagen) and 0.8 U/μL RNase inhibitor. RT reaction was carried out at48° C. in a 20.5 μL volume and the PCR primers and probes were added tothe reaction mixture in a 4.5 μL volume during a 70° C. hold followingthe RT. This was done in order to maintain PCR specificity andsensitivity. RT was tested using 30, 10, 7, 5, 3 and 2 minutes todetermine the fastest RT possible without loss of sensitivity.

Multiplex PCR Assays: In the temperature-controlled multiplex, bothprimer and probe sets for CEA and β-gus were added to the reactionmixture. In this case however, the β-gus primers (β-gus-80, 400 nmol/L)were redesigned to have a Tm of 50° C. compared to the CEA primers whichhave a Tm of 60° C. PCR was performed with a one second denaturation, afour second hold at 53° C. for annealing and a six second hold at 64° C.for extension and optical read (total anneal/extend time of tenseconds). The 53° C. anneal step was eliminated one cycle after theβ-gus amplification plot reached threshold (determined beforehand in aseparate singleplex reaction) and subsequent cycles were carried outwith only the 64° C. anneal/extend as in the optimal, rapid PCRsingleplex assay. Simple multiplexing was performed using 800 nM primersfor both genes and utilized our standard β-gus-81 (Tm 60° C.) primerset. The conventional primer-limited multiplex was carried out using 300nmol/L β-gus-81 primers since this was determined to be the lowestconcentration that did not result in a significant increase (>1 cycle)in the β-gus Ct values in a rapid PCR. The first 20 PCR cycles using theconventional methodologies were performed with an anneal/extend time of10 seconds at 64° C. followed by a change to 6 seconds for the remainingcycles. This was done in order to better simulate the conditions used inthe temperature-controlled multiplex and allow direct comparison of themethods. All three multiplex methods were tested on serial dilutions ofCEA cDNA (as described above) in order to determine the accuracy anddynamic range compared with singleplex reactions.

Internal positive controls. The internal positive controls (IPC) are DNAoligonucleotides that have the same primer sequences as the target gene(CEA or tyrosinase) but have a different internal probe sequence (Table10). Selected sites in the IPC's were synthesized with uracil instead ofthymine so that contamination with the highly concentrated mimic couldbe controlled using uracil DNA glycosylase if required. The IPCs wereadded to the reaction mastermix in amounts that were determinedempirically to give Ct values of 36-37 cycles. The rapid triplex assayswere then performed as described for the primer-limited multiplex(duplex) above with the addition of the internal control (IC) probe at aconcentration of 200 nmol/L. TABLE 10 CEA and Tyrosinase internalpositive control mimics. CEA mimic: (SEQ ID NO: 24)AGACAATCACAGTCTCTGCGGAAGCATCATCCTCTGCATGGTCAGGTCATAACTCCAAACCCGTGGAGGACAAGGAT Tyrosinase mimic: (SEQ ID NO: 25)ACTTACUCAGCCCAGCAUCATTCTAGCATCATCCTCTGCATGGTCAGGTCATTTGGAGGAGUACAACAGCCAUCAGTItalic=gene specific primer sites. Bold=probe sequence from bacterialβ-galactosidase gene.

Lymph node assays. Histologically positive and negative lymph nodes fromesophageal cancer and melanoma patients were tested using rapid,multiplexed QRT-PCR in order to demonstrate the utility of thistechnique. Lymph node RNA was analyzed using a triplex QRT-PCR (β-gus,CEA or Tyrosinase and appropriate mimic), with the temperaturecontrolled primer limiting technique. Reverse transcription was carriedout as described above with a 5 minute RT followed by a 30 second holdat 70° C. for addition of PCR primers. Cycle threshold values for β-guswere determined in a prior, singleplex run so that the temperaturechange could be initiated at the appropriate cycle in the multiplexreaction.

Results

Rapid PCR development: Rapid PCR assays were developed separately forall genes in singleplex assays prior to using them in a multiplexreaction. For development of the rapid PCR assays, the effect ofreducing both the denaturation time (10, 7, 5, 2, 1 seconds at 95° C.)and the anneal/extend time (30, 15, 13, 10, 7, 6, 3 seconds at 64° C.)was evaluated to determine any observed changes in Ct values. For thedenaturation time test the anneal/extend time was kept constant at 30seconds and for the anneal/extension time test the denaturation time waskept constant at 10 seconds. It was found that reducing the denaturationtime to 1 second had no effect on the Ct values for either CEA or β-gusgenes (FIG. 12, panel A). Similarly, reducing the annealing/extensiontime had minimal effect (˜0.5 cycles) on the Ct values for both genesdown to 5 seconds while a 3 second anneal/extend resulted in a Ctincrease of 1.1 cycles for CEA and 1.8 cycles for β-gus when comparedwith the 30 second anneal/extend control (FIG. 12, panel B). Thus, forfurther testing a 1 second denaturation and a 6 second anneal extendwere chosen. To evaluate the effect of the rapid PCR assay onsensitivity and quantitation over a wide range of CEA input amounts aserially diluted standard curve of CEA cDNA in a constant background ofβ-gus PCR target was generated. Using this standard curve, the rapidassay was compared with a more conventional PCR with 10 seconddenaturation and 30 second anneal/extension (FIG. 13). The rapid assayresulted in an average increase in Ct value of 1.6 cycles for CEA and0.2 cycles for β-gus. These increases appear to be consistent from pointto point, and as a result, the quantitative ability of the rapid assayis equal to that of the slower PCR. The total PCR time to run 40 cyclesusing the rapid assay was 15 minutes versus 48 minutes for the slowerPCR protocol.

Rapid reverse transcription: Rapid analysis of gene expression by RT-PCRrequires that both the reverse transcription and PCR components of theassay are rapid. For this reason the effect of reducing the reversetranscription time from 30 minutes down to 10, 7, 5, 3 and 2 minutes(FIG. 12, panel C) was evaluated. In this assay, the PCR times were heldconstant at 2 second denaturation and 15 second anneal/extension. Theresults showed an increase in Ct value of only 1.1 and 0.6 cycles forβ-gus and CEA respectively with a 5 minute reverse transcription and 1.1and 1.8 cycles with a 2 minute reverse transcription when compared with30 minutes. While none of these differences were significant forindividual time point comparisons, there does appear to be a clear trendtowards increased Ct with reduced time as would be expected. Thecombined effect of decreasing both the RT and PCR times on thesensitivity of the assay was evaluated using 4 different RT and PCRparameter combinations (FIG. 14). Once again a trend was observedtowards higher Ct's with shorter RT-PCR protocols but with a 20 minutetotal RT-PCR time (5 minute RT with a ⅙ PCR) the Ct difference was only1.4 cycles for both genes when compared with a 38 minute total RT-PCR(10 minute RT, 10/15 PCR).

Multiplex PCR assay: Testing of different multiplexing methods wascarried out on the same dilution series of CEA in constant background ofβ-gus that was used for the rapid PCR development. This dilution seriesspanned approximately 12 cycles, corresponding to a 4,096 folddifference in the starting CEA abundance between the first and the lastpoints on the curve (FIG. 15, panel A). The dilution series was used tocompare singleplex reactions for each gene with a simple multiplex PCR(FIG. 15, panel C), a conventional primer limited multiplex PCR (FIG.15, panel D) and our temperature controlled, primer limited multiplexPCR (FIG. 15, panel B). When a simple multiplex reaction was carried outfor both CEA and β-gus only the first three of five points from theseries reached threshold for CEA while the reaction for the moreabundant β-gus amplified consistently (not shown). Similar results wereobserved for the conventional primer limited reaction while thetemperature controlled primer limiting produced results almost identicalto the singleplex reactions.

Perhaps, more relevant than the range of CEA concentrations is the rangeof delta Ct (difference in Ct value between the more abundant β-gus geneand the CEA target gene) that can be achieved with the differenttechniques. In these experiments the β-gus Ct was constant for allassays between 19 and 20 cycles and thus the largest delta Ct in thisdilution series with a singleplex assay was almost 15 cycles (˜32,000fold difference in abundance). FIG. 16 shows the delta Ct's plotted foreach of the three multiplexing methods as well as the singleplex data.In the simple multiplex the third point in the series had a Ct value of38.5 cycles compared with 29.0 for the same point in the singleplexreaction, while subsequent points failed to amplify at all. Thus thesimple multiplex demonstrates a lack of quantitation with a true deltaCt of˜8 cycles, as well as a very limited dynamic range. Theconventional primer limited multiplex worked a little better (delta Ctfor the third dilution point was 4.0 cycles higher than the singleplex)but once again, dilution points four and five failed to amplify. Thus itseems that despite its feasibility in standard PCR reactions,conventional primer limiting is not amenable to rapid assays whereincreased primer concentrations are required. In comparison, thetemperature controlled multiplex amplified all points on the dilutionseries and no difference was observed in the delta Ct until the lastpoint. These results clearly demonstrate that the temperature controlledprimer limit was comparable to singleplex reactions in the rapid QPCRassay while all other multiplexing approaches remained inadequate.Furthermore, this technique has been utilized on other targets, such astyrosinase, and the method appears to be easily adaptable to newtargets.

Lymph node analysis using rapid, multiplex RT-PCR: For lymph nodeanalysis internal positive control mimics for CEA and tyrosinase wereincluded in the multiplex reactions. This internally controlled, rapidRT-PCR multiplex was used to evaluate lymph nodes from patients withesophageal cancer and with melanoma as well as benign lymph nodes frompatients without cancer. In three histologically positive esophagealcancer lymph nodes, high levels of CEA expression were detected. Inlymph nodes from non-cancer patients, 2 of 5 nodes had no detectablelevels of CEA expression and the remaining three had low levels of CEAexpression (FIG. 17). Furthermore, in cases where CEA signal wasdetected, the internal positive control was not amplified (FIG. 18,panel A). However, in CEA-negative samples, there was amplification ofthe CEA internal positive control confirming that negative results wereindeed due to the absence of CEA expression and not a failed CEA PCR(FIG. 18, panel B). Similar results were obtained for melanoma lymphnodes using tyrosinase mRNA as the cancer marker. Five lymph nodes thatwere histologically positive for melanoma and 5 lymph nodes without anyevidence of cancer were tested. Tyrosinase was detected at high levelsin the all histologically positive nodes and at very low levels in onlyone of the five negative nodes (data not shown). In the positive nodes,the expression of tyrosinase was higher than that of β-gus and as aresult only the tyrosinase gene amplification was seen. In all the fivenegative nodes the amplification of β-gus and the IPC was seen (FIG. 18,panels C and D).

Discussion

The introduction of rapid cycling PCR instruments capable of real timefluorescence-based quantitation has opened the door for application ofmolecular diagnostic assays in clinical situations where time is limitedor where a rapid result would be of psychological or physical benefit tothe patient. One case where rapid molecular assays may be advantageousis in intra-operative lymph node staging of malignancies such asmelanoma, breast and lung cancer. Conventional analysis of frozen tissuesections is very quick (approximately 20 minutes in most institutions)but the sensitivity of this technique for detecting small foci of tumoris relatively low. Furthermore, many patients who are staged as lymphnode negative by histologic examination suffer recurrence of theirdisease, probably as a result of occult disseminated tumor cells thatwere missed by routine examination. There is much evidence that RT-PCR,and particularly quantitative RT-PCR, can detect these occult cells andidentify patients at high risk for recurrence. If this information canbe obtained intraoperatively, surgeons will be able to make moreinformed treatment decisions regarding the need for adjuvant therapy andextent of resection.

It has been shown that it is feasible to carry out QRT-PCR in anintraoperative time frame and that this technique can be superior tofrozen sectioning and, at least, comparable to formalin fixed histology.In the present Example, these rapid QRT-PCR protocols are tested and itis shown that this complete assay can be carried out in under 20minutes. While this time frame is adequate for intra-operative testingthere are still several practical and technical hurdles that need to beaddressed before rapid QRT-PCR can be considered feasible in theclinical diagnostic setting. It is believed that the assays must bestandardized to allow data comparison between sites in multi-centertrials, automated (including RNA isolation) to eliminate operatordependent variability and contamination and controlled for all aspectsof the assay including internal endogenous and exogenous positivecontrols.

The latter of these requirements, quality controls, necessitates theincorporation of multiplexing into the rapid QRT-PCR. At least twocontrol reactions are required, an endogenous control gene to verifypresence and adequate quantity and quality of RNA and an exogenouspositive control to verify that the target gene QRT-PCR functioned withadequate sensitivity in samples that are negative for target geneexpression. Together these two controls ensure that a negative result istruly negative and not a result of failed RT-PCR. Thus, at least atriplex PCR reaction is needed. Unfortunately, maintaining quantitationin a multiplex PCR has not been easy even in a standard, slow PCR assay.This is because accumulation of one PCR product (from the most abundanttarget gene) inhibits amplification of other targets.

Initially this inhibition is seen as a shift of the cycle thresholdvalue to a higher cycle number, and a drop in the total fluorescence,when compared with a singleplex reaction (FIG. 15 panels A, C and D). Ifthe difference in abundance of the two genes is too great however, theless abundant gene will fail to amplify completely. Thus the typicalmultiplex reaction lacks quantitation and suffers from a poor dynamicrange. Inhibition of the less abundant target sequence is probably aconsequence of several factors including the accumulation ofpyrophosphate released from the addition of nucleotides during DNAsynthesis and inhibition/sequestration of the Taq enzyme by accumulatingPCR product. One way to overcome this inhibition is to somehow stopamplification of the more abundant gene soon after signal has beendetected and before pyrophosphate and PCR product have accumulatedenough to cause inhibition. Conventional methods for this rely onlimiting the primer concentration for the more abundant target gene suchthat the primers are used up, and PCR of that specific target stops,before inhibition can occur. While this method works reasonably well ina slow PCR assay, reducing primer concentration in a rapid assay resultsin a lower PCR efficiency, and eventually, complete PCR failure. In ourstudies the lowest primer concentration that did not change the cyclethreshold value for β-gus was determined and tested in a conventionalprimer limited assay using our rapid PCR protocol. While this provedslightly better than multiplexing without a primer limit, quantitationand dynamic range were still poor compared with singleplex reactions. Inthe method described herein, the melting temperature (Tm) of the primersfor the more abundant species (β-gus) are designed to function at alower temperature than those for the target gene (CEA or Tyrosinase).The PCR itself is carried out in two stages. The annealing step for thefirst stage of the PCR is performed at a relatively low annealingtemperature (53° C. in this example) until the amplification curve ofthe more abundant species reaches detection threshold. Following this,the second stage of the PCR is carried out with an annealing temperaturethat is at least 10° C. higher than the Tm of the low temperatureprimers (64° C. for this example). The higher temperature of the secondstage shifts the binding state of the low temperature β-gus PCR primersto favor the single stranded state, effectively terminatingamplification of β-gus while amplification of the CEA target continuesuninhibited. Thus, it is possible to maintain the high primerconcentration needed for rapid PCR of the more abundant control gene andstill maintain quantitation over a large dynamic range of the targetgene.

In addition to developing the rapid multiplexing technology, an internalpositive control mimic is incorporated that can be used to prove thatadequate RT-PCR sensitivity was achieved in samples that are negativefor target gene expression. This mimic utilizes the same primers as thetarget gene and therefore controls for the function of theseoligonucleotides. The DNA mimic might be replaced with an RNA mimic thatalso includes the reverse transcription primer site. This will thencontrol for both the RT and PCR steps of target gene amplification. Byadding this mimic to samples at concentrations that are low enough toproduce Ct values greater than 35 cycles, one can be sure that the RNAisolation and RT-PCR reaction not only worked but that it worked wellenough to detect a very low abundance of target gene if it were present.In cases where the target gene is highly expressed, the internal mimicmay fail to amplify. This is acceptable because the mimic is onlyintended as a control for negative reactions.

Therefore, the potential use of rapid QRT-PCR for detection ofmetastatic tumor cells in lymph nodes of cancer patients has been shown.While this is a particularly exciting use of rapid molecular diagnosticsit is only one potential application. As new cancer markers aredeveloped it is possible that rapid QRT-PCR could also be usedintraoperatively for analysis of surgical margins, in the clinic foranalysis of biopsies or fine needle aspirates or for analysis of primarytumors to predict response to chemotherapy or new biotherapies such astyrosine kinase inhibitors. Although the dual temperature PCR cyclingrequired for the temperature controlled multiplex adds approximately 2minutes to the overall reaction time, a complete, multiplexed QRT-PCRassay can still be carried out in under 22 minutes. With the addition ofexternal cooling to the SmartCycler™, or its future generations, it isbelieve that an extra 3-4 minutes can be saved. Also, in the currentwork, the cycle number at which the temperature change needed to beinitiated was pre-determined. Ultimately, a minor modification in theinstrument's software will automatically increase the PCR annealingtemperature after the endogenous control gene fluorescence reachesthreshold. Since all PCR sites are independently controlled, this willallow multiple reactions to be run at the same time with unknown amountsof starting RNA template. Finally, rapid QRT-PCR is now becomingfeasible in a clinical setting. Using technology currently underdevelopment, RNA isolation can also be carried out very quickly in anautomated process. To this end, certain fully integrated RNA isolation,reverse transcription and quantitative PCR instruments, such asCepheid's GenXpert™, will automate, according to a series of programmingsteps, all of these processes and provide a QRT-PCR result in under 30minutes. The combination of the rapid, internally controlled multiplextechnology described herein with an automated sample processing platformwill allow for accurate and yet expeditious measurements in a closedenvironment. The simplicity and sensitivity of this integrated systemshould enable many new applications of molecular assays in cancerdiagnostics.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, the disclosure of these ranges is intended as a continuous rangeincluding every value between the minimum and maximum values.

1-66. (canceled)
 67. An oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35, and a derivative thereof. 68-77. (canceled)
 78. The oligonucleotide of claim 67, wherein the derivative is capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 79. The oligonucleotide of claim 78, consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 80. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 6. 81. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 7. 82. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 13. 83. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 14. 84. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 16. 85. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 17. 86. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 19. 87. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 20. 88. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 23. 89. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 24. 90. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 25. 91. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 26. 92. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 27. 93. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 28. 94. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 29. 95. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 30. 96. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 31. 97. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 32. 98. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 33. 99. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 34. 100. The oligonucleotide of claim 78, consisting of SEQ ID NO:
 35. 101. A cartridge for use in a PCR system, the cartridge comprising one or more compartments containing a first PCR primer set and a second PCR primer set, wherein the first PCR primer set has a first effective Tm and the second PCR primer set has a second effective Tm that is different from the first effective Tm.
 102. The cartridge of claim 101, further comprising a third compartment including one of reverse transcription reagents, cell lysis reagents and RNA purification reagents.
 103. The cartridge of claim 102, comprising reverse transcriptase and a reverse transcriptase primer.
 104. The cartridge of claim 103, comprising random hexanucleotide reverse transcriptase primers.
 105. The cartridge of claim 103, comprising sequence-specific reverse transcriptase primers.
 106. The cartridge of claim 105, wherein the sequence-specific reverse transcriptase primers are specific to one or more of β-glucuronidase mRNA, carcinoembryonic antigen mRNA, tyrosinase mRNA and 18S ribosomal RNA.
 107. The cartridge of claim 101, comprising a PCR primer set for producing one or more of β-glucuronidase, carcinoembryonic antigen, tyrosinase mRNA and 18S ribosomal RNA-specific amplicon.
 108. The cartridge of claim 101, further comprising an internal positive control nucleic acid.
 109. The cartridge of claim 108, wherein the internal positive control nucleic acid comprises one or more uracil residues.
 110. The cartridge of claim 101, further comprising a fluorescent reporter for indicating accumulation of a specific amplicon in a PCR reaction.
 111. The cartridge of claim 110, wherein the fluorescent reporter is a probe for a fluorescent 5′ nuclease assay.
 112. The cartridge of claim 110, wherein the fluorescent reporter is a molecular beacon probe.
 113. The cartridge of claim 101, wherein the cartridge is disposable after a single use.
 114. The cartridge of claim 101, comprising an oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35 or a derivative thereof capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 115. The cartridge of claim 114, comprising one or more oligonucleotides consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 116. The cartridge of claim 101, wherein the effective Tm of the first primer set and the effective Tm of the second primer set differ by at least 5° C.
 117. A kit comprising a first PCR primer set and a second PCR primer set, wherein the first PCR primer set has a first effective Tm and the second PCR primer set has a second effective Tm that is different from the first effective Tm.
 118. The kit of claim 117, comprising a cartridge for use in a PCR system, the cartridge comprising one or more compartments containing the first PCR primer set and the second PCR primer set.
 119. The kit of claim 117, further comprising one of reverse transcription reagents, cell lysis reagents and RNA purification reagents.
 120. The kit of claim 119, comprising reverse transcriptase and a reverse transcriptase primer.
 121. The kit of claim 120, comprising random hexanucleotide reverse transcriptase primers.
 122. The kit of claim 120, comprising sequence-specific reverse transcriptase primers.
 123. The kit of claim 122, wherein the sequence-specific reverse transcriptase primers are specific to one or more of β-glucuronidase mRNA, carcinoembryonic antigen mRNA, tyrosinase mRNA and 18S ribosomal RNA.
 124. The kit of claim 117, comprising a PCR primer set for producing one or more of β-glucuronidase, carcinoembryonic antigen, tyrosinase mRNA and 18S ribosomal RNA-specific amplicon.
 125. The kit of claim 117, further comprising an internal positive control nucleic acid.
 126. The kit of claim 125, wherein the internal positive control nucleic acid comprises one or more uracil residues.
 127. The kit of claim 117, further comprising a fluorescent reporter for indicating accumulation of a specific amplicon in a PCR reaction.
 128. The kit of claim 127, wherein the fluorescent reporter is a probe for a fluorescent 5′ nuclease assay.
 129. The kit of claim 127, wherein the fluorescent reporter is a molecular beacon probe.
 130. The kit of claim 117, comprising an oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35 or a derivative thereof capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 131. The kit of claim 130, comprising one or more oligonucleotides consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
 132. The kit of claim 117, wherein the effective Tm of the first primer set and the effective Tm of the second primer set differ by at least 5° C. 